Chimeric Polypeptides and Methods of Use

Abstract

Aspects of the present disclosure provide compositions and methods for detecting, isolating, depleting and / or purifying cells comprising polypeptides, including chimeric polypeptides, useful in such methods. Various chimeric polypeptides are disclosed, along with the use of such polypeptides as selection markers, transduction markers and / or safety switches. Cells, including therapeutic cells such as T cells, NK cells, NKT cells and iPSCs, that comprise a polynucleotide encoding one or more chimeric polypeptides are also disclosed.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 188,936, filed May 14, 2021, and U.S. Provisional Patent Application No. 63 / 274,765, filed November 2, 2021, both of which are incorporated by reference in their entireties herein.

[0002] Aspects of the present disclosure relate to the field of molecular biology. In particular, embodiments of the present invention relate to chimeric polypeptides, engineered cells and methods of use thereof. [Background technology]

[0003] Therapeutic cells targeting tumor antigens, such as CAR-NK cells, CART cells and TCR-transduced T cells, are promising approaches for the treatment of various malignancies. Despite the success of these therapies to date, several limitations remain, including disease recurrence, high manufacturing costs, and toxicity. There is a need for compositions and methods for targeting therapeutic cells, such as for isolation, identification and purification during manufacturing, and for use in selectively targeting such cells for elimination in the case of adverse events, such as cytokine release syndrome and graft-versus-host disease. Summary of the Invention

[0004] In some aspects, methods and compositions are disclosed herein that are useful in detecting, isolating, depleting and / or purifying cells. Thus, in some embodiments, chimeric polypeptides are disclosed that include one or more extracellular domains (e.g., from BCMA, Trop2, CD30, EGFR or Her2) and a transmembrane domain. In some embodiments, the chimeric polypeptides further include one or more additional domains, such as a signal peptide, a hinge region or an intracellular domain. Engineered cells that express such polypeptides, as well as methods for detecting, isolating, depleting and / or purifying such cells, are also disclosed.

[0005] Embodiments of the present disclosure include nucleic acids, polynucleotides, polypeptides, proteins, peptides, constructs, vectors, cells, therapeutic cells, immune cells, engineered cells, methods of making engineered cells, methods of detecting engineered cells, methods of isolating engineered cells, methods of depleting engineered cells, and methods of purifying engineered cells. Nucleic acids of the present disclosure may encode one or more polypeptides of the present disclosure, including one or more chimeric polypeptides. In some embodiments, nucleic acid molecules of the present disclosure encode chimeric polypeptides. In some embodiments, nucleic acid molecules of the present disclosure encode two or more chimeric polypeptides. Chimeric polypeptides of the present disclosure can include at least one, two, three, or more of the following regions or domains: signal peptide, extracellular domain, hinge region, transmembrane domain, and intracellular region. Engineered cells of the present disclosure can include one, two, three, four, or more polynucleotides and / or polypeptides of the present disclosure. The methods of the disclosure may include at least one, two, three, four, or more of the following steps: introducing a polynucleotide into a cell, introducing a vector into a cell, introducing a polypeptide into a cell, expressing the polypeptide in the cell, expanding a population of cells, contacting the cells with an antigen binding protein, contacting the cells with an antibody drug conjugate, and detecting the cells with an imaging agent.

[0006] Disclosed herein, in some embodiments, is a chimeric polypeptide comprising: (a) an extracellular domain derived from B-cell maturation antigen (BCMA); (b) a hinge region derived from programmed cell death 1 ligand 1 (PDL1); (c) a transmembrane domain; and (d) an intracellular region. In some embodiments, the extracellular domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19. In some embodiments, the extracellular domain comprises SEQ ID NO: 19. In some embodiments, the extracellular domain consists of SEQ ID NO: 19. In some embodiments, the hinge region comprises SEQ ID NO: 23. In some embodiments, the hinge region consists of SEQ ID NO: 23. In some embodiments, the transmembrane domain is a transmembrane domain from the alpha or beta chain of the T cell receptor, or CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:26. In some embodiments, the transmembrane domain comprises SEQ ID NO:26. In some embodiments, the transmembrane domain consists of SEQ ID NO:26. In some embodiments, the transmembrane domain is a transmembrane domain from PDL1. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.In some embodiments, the transmembrane domain comprises SEQ ID NO:25. In some embodiments, the transmembrane domain consists of SEQ ID NO:25. In some embodiments, the intracellular region comprises the sequence RLR (SEQ ID NO:29). In some embodiments, the intracellular region consists of the sequence RLR (SEQ ID NO:29). In some embodiments, the intracellular region comprises SEQ ID NO:31. In some embodiments, the intracellular region comprises SEQ ID NO:31. In some embodiments, the intracellular region comprises SEQ ID NO:32. In some embodiments, the intracellular region consists of SEQ ID NO:32. In some embodiments, the intracellular region comprises an amino acid sequence at least 95% identical to SEQ ID NO:40. In some embodiments, the intracellular region comprises SEQ ID NO:40. In some embodiments, the intracellular region consists of SEQ ID NO:40. In some embodiments, the intracellular region comprises at most 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In some embodiments, the chimeric polypeptide is 100 amino acids in length or less. In some embodiments, the chimeric polypeptide does not include a signaling domain, hi some embodiments, the chimeric polypeptide does not include an intracellular region from BCMA.

[0007] In some embodiments, the chimeric polypeptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In some embodiments, the chimeric polypeptide comprises SEQ ID NO: 1. In some embodiments, the chimeric polypeptide consists of SEQ ID NO: 1. In some embodiments, the chimeric polypeptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 38. In some embodiments, the chimeric polypeptide comprises SEQ ID NO: 38. In some embodiments, the chimeric polypeptide consists of SEQ ID NO: 38. Also disclosed are nucleic acid molecules comprising a nucleotide sequence encoding a chimeric polypeptide. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2. In some embodiments, the nucleic acid molecule comprises SEQ ID NO:2. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 2. Vectors comprising the nucleic acid molecules are also disclosed.

[0008] Also disclosed herein, in some embodiments, is a chimeric polypeptide comprising: (a) a tissue-type plasminogen activator (tPA) signal peptide; (b) an extracellular domain from BCMA; (c) a hinge region; (d) a transmembrane domain; and (e) an intracellular region. In some embodiments, the tPA signal peptide comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to SEQ ID NO:34. In some embodiments, the tPA signal peptide comprises SEQ ID NO:34. In some embodiments, the tPA signal peptide consists of SEQ ID NO:34. In some embodiments, the extracellular domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19. In some embodiments, the extracellular domain comprises SEQ ID NO: 19. In some embodiments, the extracellular domain consists of SEQ ID NO: 19. In some embodiments, the hinge region comprises a CD8α hinge, a PDL1 hinge, an IgG4 hinge, an IgG1 hinge, or a CD34 hinge. In some embodiments, the hinge region is a hinge region from PDL1. The hinge region comprises SEQ ID NO: 23. In some embodiments, the hinge region consists of SEQ ID NO: 23. In some embodiments, the hinge region is a hinge region from CD8α. In some embodiments, the hinge region comprises SEQ ID NO: 24. In some embodiments, the hinge region consists of SEQ ID NO: 24. In some embodiments, the transmembrane domain is a transmembrane domain from the alpha or beta chain of the T cell receptor, or CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154.In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26. In some embodiments, the transmembrane domain comprises SEQ ID NO: 26. In some embodiments, the transmembrane domain consists of SEQ ID NO: 26. In some embodiments, the intracellular region is a portion of the intracellular region from CD8α. In some embodiments, the intracellular region comprises at most 10, 9, 8, 7 or 6 amino acids. In some embodiments, the intracellular region comprises SEQ ID NO: 30. In some embodiments, the intracellular region consists of SEQ ID NO: 30. In some embodiments, the chimeric polypeptide is 150 amino acids in length or less. In some embodiments, the chimeric polypeptide does not comprise a signaling domain. In some embodiments, the chimeric polypeptide does not include an intracellular region from BCMA. In some embodiments, the chimeric polypeptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3. In some embodiments, the chimeric polypeptide comprises SEQ ID NO:3. In some embodiments, the chimeric polypeptide consists of SEQ ID NO:3. Nucleic acid molecules comprising a nucleotide sequence encoding the chimeric polypeptide are also disclosed. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 4. Also disclosed are vectors comprising the nucleic acid molecule.

[0009] Further disclosed herein, in some embodiments, is a chimeric polypeptide comprising: (a) an extracellular domain derived from CD30; (b) a transmembrane domain derived from CD30; and (c) an intracellular region derived from BCMA. In some embodiments, the extracellular domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20. In some embodiments, the extracellular domain comprises SEQ ID NO:20. In some embodiments, the extracellular domain consists of SEQ ID NO:20. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:27. In some embodiments, the transmembrane domain comprises SEQ ID NO:27. In some embodiments, the transmembrane domain consists of SEQ ID NO:27. In some embodiments, the intracellular region comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some embodiments, the intracellular region comprises SEQ ID NO: 33. In some embodiments, the intracellular region consists of SEQ ID NO: 33. In some embodiments, the chimeric polypeptide does not comprise a signaling domain. In some embodiments, the chimeric polypeptide does not comprise an intracellular region from CD30. In some embodiments, the chimeric polypeptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. In some embodiments, the chimeric polypeptide comprises SEQ ID NO: 9. Nucleic acid molecules comprising a nucleotide sequence encoding the chimeric polypeptide are also disclosed.In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 10. Also disclosed are vectors comprising the nucleic acid molecule.

[0010] Also disclosed herein in some embodiments is a chimeric polypeptide comprising (a) a signal peptide that is not a Her2 signal peptide, (b) an extracellular domain from Her2, and (c) a transmembrane domain from Her2. In some embodiments, the chimeric polypeptide does not comprise an intracellular region. In some embodiments, the signal peptide is a signal peptide from CD8α. In some embodiments, the signal peptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:35. In some embodiments, the signal peptide comprises SEQ ID NO:35. In some embodiments, the signal peptide consists of SEQ ID NO:35. In some embodiments, the extracellular domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21. In some embodiments, the extracellular domain comprises SEQ ID NO:21. In some embodiments, the extracellular domain consists of SEQ ID NO:21. In some embodiments, the transmembrane domain comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:28. In some embodiments, the transmembrane domain comprises SEQ ID NO:28. In some embodiments, the transmembrane domain consists of SEQ ID NO: 28. In some embodiments, the chimeric polypeptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11. In some embodiments, the chimeric polypeptide comprises SEQ ID NO: 11. In some embodiments, the chimeric polypeptide consists of SEQ ID NO: 11.Also disclosed are nucleic acid molecules comprising a nucleotide sequence encoding the chimeric polypeptide. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 12. Also disclosed are vectors comprising the nucleic acid molecule.

[0011] Further disclosed herein, in some embodiments, is an engineered immune cell comprising a nucleic acid encoding a Trop2 polypeptide. In some embodiments, the Trop2 polypeptide comprises SEQ ID NO: 15. In some embodiments, the nucleic acid comprises a nucleic acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the nucleic acid comprises SEQ ID NO: 16.

[0012] Also disclosed herein in some embodiments is a chimeric polypeptide comprising: (a) a signal peptide; (b) an extracellular region comprising (i) EGFR domain III and (ii) a portion of EGFR domain IV having a length of less than 100 amino acids; (c) a hinge region; and (d) a transmembrane domain. In some embodiments, the portion of EGFR domain IV has a length of less than 75, 70, 65, 60, 55, 50, 45, 40, or 35 amino acids. In some embodiments, the portion of EGFR domain IV has a length of 33 amino acids. In some embodiments, the signal peptide is a signal peptide derived from GM-CSFRα. In some embodiments, the signal peptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:36. In some embodiments, the signal peptide comprises SEQ ID NO:36. In some embodiments, the signal peptide consists of SEQ ID NO:36. In some embodiments, the extracellular region comprises an amino acid sequence at least 95% identical to SEQ ID NO:22. In some embodiments, the extracellular region comprises SEQ ID NO:22. In some embodiments, the extracellular region consists of SEQ ID NO:22. In some embodiments, the hinge region is a hinge region from CD8. In some embodiments, the hinge region comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37. In some embodiments, the hinge region comprises SEQ ID NO:37. In some embodiments, the hinge region consists of SEQ ID NO:37. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26. In some embodiments, the transmembrane domain comprises SEQ ID NO:26.In some embodiments, the transmembrane domain consists of SEQ ID NO:26. In some embodiments, the chimeric polypeptide does not include a signaling domain. In some embodiments, the chimeric polypeptide does not include an intracellular region from EGFR. In some embodiments, the chimeric polypeptide comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:15. In some embodiments, the chimeric polypeptide comprises SEQ ID NO:15. In some embodiments, the chimeric polypeptide consists of SEQ ID NO:15. Nucleic acid molecules comprising a nucleotide sequence encoding the chimeric polypeptide are also disclosed. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 16. Also disclosed are vectors comprising the nucleic acid molecule.

[0013] In some embodiments, a method of making an engineered cell is disclosed that comprises introducing into a cell a chimeric polypeptide, a nucleic acid molecule, or a vector of the present disclosure.

[0014] In some embodiments, engineered cells are disclosed that contain a nucleic acid molecule encoding a chimeric polypeptide of the present disclosure. In some embodiments, the engineered cell is a T cell. In some embodiments, the T cell is a CD4 + T cells, CD8 +The engineered cell is a T cell, an iNKT cell, an NKT cell, a gamma delta T cell, or a regulatory T cell. In some embodiments, the engineered cell is a natural killer (NK) cell. In some embodiments, the engineered cell is an induced pluripotent stem cell (iPSC). In some embodiments, the engineered cell is an iPSC-derived cell. In some embodiments, the engineered cell further comprises a chimeric antigen receptor (CAR). In some embodiments, the chimeric polypeptide is operably linked to the CAR. In some embodiments, the engineered cell further comprises a T cell receptor (TCR). In some embodiments, the chimeric polypeptide is operably linked to the TCR. Populations of cells comprising the engineered cells of the present disclosure are also disclosed.

[0015] Further disclosed are methods for detecting, isolating, depleting or purifying engineered cells of the present disclosure, comprising contacting the engineered cells with an antigen binding protein, wherein the antigen binding protein is capable of binding to a polypeptide of the engineered cells. In embodiments where the engineered cells express a chimeric polypeptide comprising an extracellular domain from BCMA, the antigen binding protein is a BCMA binding protein. In embodiments where the engineered cells express a chimeric polypeptide comprising an extracellular domain from CD30, the antigen binding protein is a CD30 binding protein. In embodiments where the engineered cells express a chimeric polypeptide comprising an extracellular domain from Her2, the antigen binding protein is a Her2 binding protein. In embodiments where the engineered cells express a Trop2 polypeptide, the antigen binding protein is a Trop2 binding protein. In embodiments where the engineered cells express a chimeric polypeptide comprising an extracellular region comprising a portion of the extracellular domain from EGFR, the antigen binding protein is an EGFR binding protein. In some embodiments, the antigen binding protein is an antigen-specific antibody or an antigen-binding fragment thereof. In some embodiments, the antigen binding protein is linked to an imaging agent and the method further comprises detecting the cell with the imaging agent. In some embodiments, the antigen binding protein is linked to a cytotoxic agent (e.g., an antibody-drug conjugate). In some embodiments, contacting the engineered cell with the antigen binding protein is performed in vitro. In some embodiments, contacting the engineered cell with the antigen binding protein is performed ex vivo. In some embodiments, contacting the engineered cell with the antigen binding protein is performed in vivo.

[0016] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the measuring or quantification method.

[0017] The use of the words "a" or "an" when used in conjunction with the term "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0018] The phrase "and / or" means "and" or "or." By way of example, A, B, and / or C includes A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination. In other words, "and / or" operates as an inclusive or.

[0019] The terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (any form of having, such as "have" and "has"), "including" (any form of including, such as "includes" and "include") or "containing" (any form of including, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The compositions and methods of their use can "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the scope of the claims to particular materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0021] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0022] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as the "use" of any compound, composition, or agent discussed herein to achieve or carry out the stated therapeutic, diagnostic, or physiological purpose or effect.

[0023] The term "engineered" as used herein refers to an entity produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered entity is synthetic and contains elements that do not occur in nature or are not constructed as utilized in the present disclosure. In certain embodiments, the vector is engineered by recombinant nucleic acid techniques and the cell is engineered by transfection or transduction of the engineered vector.

[0024] As used herein, "prevent" and similar words such as "prevented," "preventing," and the like refer to an approach to prevent, inhibit, or reduce the likelihood of onset or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0025] The term "subject" as used herein generally refers to an individual having a biological sample undergoing processing or analysis, and in certain cases, having or suspected of having cancer. A subject may be any living organism or animal subject that is the subject of a method or material, including mammals, for example, humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject may be a patient, and may have or be suspected of having a disease (which may be referred to as a medical condition), such as, for example, a benign or malignant neoplasm or cancer. A subject may be undergoing or have undergone a treatment. A subject may be asymptomatic. A subject may be a healthy individual, but an individual who desires to prevent cancer. The term "individual" may be used interchangeably, at least in some cases. As used herein, a "subject" or "individual" may or may not be housed in a medical facility, and may be treated as an outpatient in a medical facility. An individual may receive one or more pharmaceutical compositions via the Internet. An individual may include a human or non-human animal of any age, and thus includes both adults and juveniles (i.e., children) and infants, including intrauterine individuals. The term is not intended to imply a need for medical treatment, and thus an individual may be a part of an experiment, whether clinical or in support of basic science research, either voluntarily or involuntarily.

[0026] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal decrease in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may include either a reduction or amelioration of symptoms of the disease or condition, or a delay in the progression of the disease or condition, as the case may be. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0027] It is specifically contemplated that any limitation described with respect to one embodiment of the present invention may be applied to any other embodiment of the present invention. Moreover, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention. Any embodiment described with respect to one aspect of the present disclosure may also be applied to other aspects of the present disclosure, and vice versa. For example, any step in a method described herein may be applied to any other method. Moreover, any method described herein may exclude any step or combination of steps. Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments described elsewhere in different examples or elsewhere in this application, such as in the Abstract, Detailed Description, Claims, and Brief Description of the Figures.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention: Embodiments of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] [Figure 1] Shown is GFP expression and BCMA staining of 293T cells transfected with plasmids containing one of three different membrane bound receptors, the BCMA extracellular domain fused to the PD-L1 hinge and transmembrane domain, the BCMA extracellular domain fused to the CD8α hinge and transmembrane domain, or full length wild type BCMA.

[0031] [Diagram 2] Shown are CD19CAR and BCMA staining of primary T cells (transduced with BCL6 and BCL2L1) transduced with lentiviral vectors co-expressing CD19CAR and one of two different BCMA fusion constructs, the BCMA extracellular domain with the PD-L1 hinge and transmembrane domain but without a functional intracellular domain (left panel), or the BCMA extracellular domain fused to the PD-L1 hinge and transmembrane domain and the cytoplasmic domain from Trop-2 (tBCMA; middle panel). An unstained control is shown in the right panel.

[0032] [Diagram 3] Shown is CD19CAR and BCMA staining of cells enriched using anti-APC magnetic beads.

[0033] [Figure 4] Shown is the percent viable cell change of primary T cells transduced with BCMA fusion proteins (transduced with BCL6 and BCL2L1) treated with the indicated concentrations of belantamab mafodotin.

[0034] [Diagram 5] Shown is BCMA staining (horizontal axis) of Jurkat cells transduced with lentiviral vectors expressing either the BCMA ectodomain alone, the BCMA ectodomain fused to the cytoplasmic domain from CD317, or the BCMA ectodomain fused to the cytoplasmic domain from CD3γ, and treated with the indicated concentrations of belantamab mafodotin (0, 12.5 or 25 μg).

[0035] [Figure 6] CD30 and CD69 staining of primary T cells transfected with BCL6 and BCL2L1 is shown.

[0036] [Figure 7] Figure 1 shows the percent viable cells of BCL6- and BCL2L1-transfected primary T cells treated with the indicated concentrations of brentuximab vedotin for the indicated times.

[0037] [Figure 8] A schematic diagram of the construct encoding both the CD19CAR and 1) Her2 domain 4 and 2) the CD30 extracellular domain fused to the BCMA cytoplasmic tail is shown.

[0038] [Figure 9] Shown are CD30 (left panel) and Her2 (middle panel) staining of 293T cells transduced with the constructs shown in Figure 8. The right panel shows the change in viable cell counts after 4 days of treatment with the indicated concentrations of brentuximab.

[0039] [Figure 10] A schematic diagram of the constructs encoding CD19CAR and both 1) Her2 domain 4 and 2) truncated EGFR is shown.

[0040] [Figure 11A] Her2 and CD19CAR staining of Jurkat T cells transduced with the constructs shown in FIG. 10 are shown. [Figure 11B] Shown is the percent viable cell change following treatment of cells with the indicated concentrations of trastuzumab (Herceptin®).

[0041] [Figure 12] Schematic diagram of the construct encoding CD19CAR and both 1) Her2 domain 4 and 2) Trop2.

[0042] [Figure 13] Shown is CD19CAR and Trop2 staining of 293T cells transfected with the constructs shown in FIG.

[0043] [Figure 14] A schematic diagram of the constructs encoding CD19CAR and both 1) Her2 domain 4 and 2) truncated EGFR is shown.

[0044] [Figure 15] Shown is CD19CAR and EGFR staining (cetuximab) of 293T cells transfected with the constructs shown in FIG.

[0045] [Figure 16A] tBCMA safety switch with in vitro efficacy. T cells transfected with the tBCMA safety switch, BCL6 and BCL2L1 are the cluster in the left bar, T cells transfected with the tBCMA safety switch, CD19CAR, BCL6 and BCL2L1 are the cluster in the middle bar, and untransfected control Raji cells are the cluster in the right bar.

[0046] [Figure 16B] 1 shows a tBCMA safety switch with in vivo efficacy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Aspects of the present disclosure relate to polypeptides useful for the detection, isolation, depletion and / or purification of cells. Thus, certain aspects of the present disclosure relate to chimeric polypeptides comprising, for example, an extracellular domain from BCMA, an extracellular domain from Trop-2, an extracellular domain from CD30, an extracellular domain from EGFR (e.g., a portion of domain III and domain IV from EGFR) and / or an extracellular domain from Her2 (e.g., domain IV from HER2). The chimeric polypeptides of the present disclosure may also comprise one or more additional domains or regions, such as a signal peptide, a hinge, a transmembrane region and / or one or more intracellular regions. Also disclosed are cells (e.g., therapeutic cells) comprising one or more polypeptides (e.g., chimeric polypeptides) of the present disclosure, as well as methods for detecting, isolating, depleting and / or purifying such cells.

[0048] In certain embodiments, the chimeric polypeptides are utilized for one or more specific purposes, for example, related to cell therapy. In certain embodiments, the chimeric polypeptides are utilized for direct or indirect control of the use of specific therapeutic cells, allowing for monitoring of cell therapy, detection of cells in cell therapy, isolation of cells for cell therapy, and / or termination of cell therapy at a desired event and / or time. In certain embodiments, the chimeric polypeptides are utilized as transduction markers, safety switches, or both. In some cases, the chimeric polypeptides encompassed herein are used as transduction markers or selection markers in cells, while another safety switch controls inhibition of the cells. In other cases, the chimeric polypeptides are used as safety switches in cells, optionally in addition to another safety switch in the same cell.

[0049] The safety switch chimeric polypeptides can be used in transduced / transfected cells to induce cell death as needed. Cells utilizing the chimeric polypeptides can contain one or more different chimeric polypeptides that are safety switches. In some embodiments, the safety switch chimeric polypeptides are utilized as "suicide genes" that result in the transition of the gene product to a compound that will kill the host cell upon administration of a prodrug or other agent. In other embodiments, the safety switch chimeric polypeptides are utilized as suicide genes that encode a gene product that can be targeted, if desired, by an agent (e.g., an antibody) that targets the suicide gene product.

[0050] In some cases, an individual undergoes cell therapy in which the cells express the chimeric polypeptide. If an individual undergoing cell therapy and / or having undergone cell therapy is considered to be at risk of having one or more symptoms of one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (for example), the individual may be subjected to the use of one or more agents that bind to the extracellular domain of the chimeric polypeptide. The use of agents that bind to the chimeric polypeptide may be part of a planned protocol for treatment, or may be used only when there is a recognized need for its use. In some cases, cell therapy is terminated by the use of agents that target the extracellular domain of the chimeric polypeptide, since treatment is no longer required.

[0051] The use of the chimeric polypeptide as a safety switch may be initiated upon the onset of at least one adverse event to an individual, which may be recognized by any means, including upon routine monitoring, which may or may not be continuous from the start of cell therapy. The adverse event may be detected upon examination and / or testing. If an individual has cytokine release syndrome (which may also be referred to as cytokine storm), the individual may have elevated levels of inflammatory cytokines (e.g., interferon-gamma, granulocyte macrophage colony stimulating factor, IL-10, IL-6, and TNF-alpha, by way of example only), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leakage, cardiac / renal / hepatic dysfunction, or a combination thereof. If an individual has neurotoxicity, the individual may have confusion, delirium, aphasia, and / or seizures. In some cases, the individual is tested for markers associated with the onset and / or severity of cytokine release syndrome (e.g., C-reactive protein, IL-6, TNF-alpha, and / or ferritin, etc.).

[0052] In certain embodiments, cell therapy may include one or more vectors encoding one or more heterologous proteins, and such heterologous proteins may be compositions that render the cells therapeutic. In some embodiments, the vector encoding one or more heterologous proteins encodes one or more safety switches. The safety switch may or may not be on the same vector as the CAR, for example. If the safety switch is on the same vector as the CAR, the safety switch and the CAR may be separated, for example, by an IRES or 2A element.

[0053] I. Polypeptides Aspects of the present disclosure relate to polypeptides, including chimeric polypeptides, and methods of using the same. As used herein, "protein" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to an endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is used, while in many embodiments of the present disclosure, a modified protein or polypeptide is used. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "mutant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild type protein or polypeptide. In some embodiments, a modified / mutated protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / mutated protein or polypeptide may be altered with respect to one activity or function, but retains the wild type activity or function in other respects.

[0054] When a protein is specifically mentioned herein, it is generally a reference to a natural (wild type) or recombinant (modified) protein, or a protein with any signal sequence optionally removed. A protein can be directly isolated from an organism in which it is naturally occurring, can be produced by recombinant DNA / exogenous expression methods, or can be produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant" may be used in conjunction with a polypeptide or the name of a particular polypeptide, which generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a product of replication of such a molecule.

[0055] In certain embodiments, the size of a protein or polypeptide (wild type or modified) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 9, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 85 The term "domain" may include, but is not limited to, 0, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any derivable range therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that the polypeptides can be mutated by truncation to make them shorter than their corresponding wild-type forms, and can also be altered by fusing or conjugating heterologous proteins or polypeptide sequences with specific functions (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids having a structure or function recognizable by one of skill in the art.

[0056] A polypeptide, protein, or a polynucleotide encoding such a polypeptide or protein of the present disclosure may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acid or nucleic acid substitutions, or may be a variant of the sequence 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable extent therein) similar to, identical to, or at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 9, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 , 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 21 5, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleotides, or any range derivable therein.

[0057] In some embodiments, the protein, polypeptide, or nucleic acid is selected from the group consisting of 1-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109, 110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,1 41,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,1 72,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,20 3,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234 ,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,79 6,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,8 27,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857 ,858,859,860,861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,88 8,889,890,891,892,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918, 919,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949 ,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966,967,968,969,970,971,972,973,974,975,976,977,978,979,980,981,982,983,984,985,986,987,988,989,990,991,992,993,994,995,996,997,998, 999, or 1000 (or any derivable range therein) amino acids or nucleotides.

[0058] In some embodiments, the protein, polypeptide, or nucleic acid is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109, 110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,1 41,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,1 72,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,20 3,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234 ,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,79 6,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,8 27,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857, 858,859,860,861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888 ,889,890,891,892,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918,91 9,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949,9 99, 998, 999, 1000, 991, 992, 993, 994, 995, 996, 997, 998, 999, 10 ...

[0059] In some embodiments, the polypeptide, protein, or nucleic acid is at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to one of SEQ ID NOs: 1-48, respectively. Similar, identical, or homologous to SEQ ID NOs: 1 to 48, which are at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,1 12,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,14 3,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174 ,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,796,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,827,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888,889,890,891,892,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918,919,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) consecutive amino acids or nucleotides.

[0060] In some embodiments, any one of SEQ ID NOs: 1-48 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 8,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114, 115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,1 46,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,1 77,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,20 8,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239 ,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,796,797,798,799,800,801,8 02,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,827,828,829,830,831,832,83 3,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864 ,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888,889,890,891,892,893,894,895, 896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918,919,920,921,922,923,924,925,926,9 27,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,95 8,959,960,961,962,963,964,965,966,967,968,969,970,971,972,973,974,975,976,977,978,979,980,981,982,983,984,985,986,987,988,989,990,991,992,993,994,995,996,997,998, 999, or 1000 and including at least, up to, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319, ,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,796,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,827,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864,865,866,867,868 ,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888,889,890,891,892,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917 ,918,919,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966 , 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range therein) consecutive amino acids or nucleotides.

[0061] Polypeptides and peptides of the disclosure may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein) or more variant amino acid substitutions, or have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 1 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein) similar, identical, or homologous to at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids or nucleotides, or any derivable range therein.

[0062] In some embodiments, a peptide, polypeptide or nucleic acid may comprise 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (or any derivable range therein) amino acids or nucleotides of any of SEQ ID NOs: 1-48.

[0063] In some embodiments, a peptide, polypeptide or nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 (or any derivable range therein) consecutive amino acids of SEQ ID NOs: 1-48.

[0064] In some embodiments, the peptide, polypeptide or nucleic acid has a sequence identical to one of SEQ ID NOs: 1-48 by at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to SEQ ID NOs: 1-48.

[0065] In some embodiments, there is a peptide, polypeptide, or nucleic acid that begins at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of any of SEQ ID NOs: 1-48 and includes, at least includes, or includes up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (or any derivable range therein) consecutive amino acids or nucleotides of any of SEQ ID NOs: 1-48.

[0066] A peptide, polypeptide or nucleic acid may comprise, at least may comprise, or may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or any derivable range therein) amino acid or nucleotide substitutions compared to SEQ ID NOs: 1-48. A peptide, polypeptide or nucleic acid may comprise, at least may comprise, or may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or any derivable range therein) amino acid or nucleotide substitutions, and the substitutions may be at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or 25 relative to SEQ ID NOs: 1-48. In the case of amino acid substitutions, the substitutions (e.g., at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or 25) can be with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In the case of nucleic acid substitutions, the substitutions (e.g., at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or 25) can be with guanine, cytosine, adenine, thymine, uracil, or other nucleotides.

[0067] Nucleotide and protein, polypeptide and peptide sequences of various genes have been previously disclosed and can be found in recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information's GENBANK® and GENPEPT® databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art.

[0068] In the compositions of the present disclosure, in some embodiments, it is contemplated that there is from about 0.001 mg to about 10 mg / ml of total polypeptide, peptide and / or protein. The concentration of protein in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any derivable range therein).

[0069] A. Chimeric Polypeptides As disclosed herein, a "chimeric polypeptide" describes any polypeptide having regions, domains, or other portions derived from two or more different polypeptides. For example, an example of a chimeric polypeptide is a polypeptide having a first domain derived from a first protein and a second domain derived from a second protein. A region or domain is described as "from" or "derived from" a protein or polypeptide if the region or domain has the same sequence as at least a portion of the polypeptide. Thus, for example, a hinge region derived from PDL1 (also "derived from PDL1") refers to a hinge region that has the same sequence as at least a portion of the hinge region of the PDL1 protein. A region or domain "derived from" a protein or polypeptide may also be described as a protein or polypeptide region or domain. For example, a transmembrane domain derived from CD30 may also be described as a "CD30 transmembrane domain". A chimeric polypeptide may include regions from at least two, three, four, five, six, or more different polypeptides. Examples of chimeric polypeptides include chimeric antigen receptors (CARs) and other chimeric cell surface polypeptides. In some embodiments, a chimeric polypeptide of the present disclosure is a polypeptide comprising an extracellular domain from BCMA and one or more additional regions or domains from a protein that is not BCMA. In some embodiments, a chimeric polypeptide of the present disclosure is a polypeptide comprising an extracellular domain from CD30 and one or more additional regions or domains from a protein that is not CD30. In some embodiments, a chimeric polypeptide of the present disclosure is a polypeptide comprising an extracellular domain from Her2 and one or more additional regions or domains from a protein that is not Her2. In some embodiments, a chimeric polypeptide of the present disclosure is a polypeptide comprising an extracellular domain from EGFR and one or more additional regions or domains from a protein that is not EGFR.Chimeric polypeptides of the present disclosure may include, for example, a signal peptide (e.g., tissue-type plasminogen activator (tPA) signal peptide, CD8α signal peptide, or GM-CSFRα signal peptide), an extracellular domain (e.g., an extracellular domain from BCMA, an extracellular domain from CD30, an extracellular domain from Her2, or an extracellular domain from EGFR), a hinge domain (e.g., a hinge domain from PDL1 or a hinge domain from CD8), a transmembrane domain (e.g., a transmembrane domain from PDL1, a transmembrane domain from CD8, a transmembrane domain from CD30, or a transmembrane domain from Her2), an intracellular region (e.g., an intracellular region from CD8, an intracellular region from CD317, an intracellular region from CD3γ, or an intracellular region from BCMA), or any combination thereof. One or more of these regions or domains may be excluded from certain embodiments of the present disclosure.

[0070] In some embodiments, disclosed herein are chimeric polypeptides comprising the extracellular domain, hinge region, transmembrane domain, and intracellular region from BCMA. The chimeric polypeptide may comprise an extracellular domain having an amino acid sequence identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to an extracellular domain from a BCMA protein. In some embodiments, the extracellular domain from BCMA is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to SEQ ID NO:19.

[0071] A chimeric polypeptide of the disclosure can include a hinge region from, for example, CD8α, PDL1, IgG4, IgG1, or CD34. In some embodiments, the hinge region has an amino acid sequence identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to a CD8α hinge, a PDL1 hinge, an IgG4 hinge, an IgG1 hinge, or a CD34 hinge. In some embodiments, the hinge region is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to the PDL1 hinge. In some embodiments, the hinge region is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to SEQ ID NO:23. In some embodiments, the hinge region comprises SEQ ID NO:23.In some embodiments, the hinge region is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to a CD8α hinge. In some embodiments, the hinge region is a hinge region from CD8α. In some embodiments, the hinge region is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to SEQ ID NO:24. In some embodiments, the hinge region is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to SEQ ID NO: 37. In some embodiments, the hinge region comprises SEQ ID NO:37.

[0072] The chimeric polypeptides of the present disclosure may include a transmembrane region from one or more transmembrane proteins. Chimeric polypeptides of the present disclosure may include, for example, 4-1BB / CD137, the alpha chain of the T cell receptor, the beta chain of the T cell receptor, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD9, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD16, CD18 (ITGB2), CD19 (B4), CD22, CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD33, CD37, CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD64, CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD80, CD84 (SLAMF5), CD86, C D96 (antennary), CD100 (SEMA4D), CD103 (ITGAE), CD123, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD154, CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (S ELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD2 76(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8),The protein may include a transmembrane domain derived from a CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, or TNF receptor protein. In some embodiments, the transmembrane domain is a transmembrane domain from the alpha or beta chain of the T cell receptor, or CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154. In some embodiments, the transmembrane domain is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to the CD8α transmembrane domain. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. In some embodiments, the transmembrane domain is identical, at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to SEQ ID NO: 26. In some embodiments, the transmembrane domain comprises SEQ ID NO: 26. In some embodiments, the transmembrane domain comprisesIt is identical, at least identical, or at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to the CD30 transmembrane domain. In some embodiments, the transmembrane domain is a transmembrane domain from CD30. In some embodiments, the transmembrane domain is identical, at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to SEQ ID NO:27. In some embodiments, the transmembrane domain comprises SEQ ID NO:27. In some embodiments, the transmembrane domain is identical, or at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical to the Her2 transmembrane domain. In some embodiments, the transmembrane domain is a transmembrane domain from Her2. In some embodiments, the transmembrane domain is identical, or at least identical, or at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 11386%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) identical. In some embodiments, the transmembrane domain comprises SEQ ID NO:28.

[0073] The chimeric polypeptides of the present disclosure may comprise an intracellular region (also "cytoplasmic region") from a transmembrane protein or proteins. As used herein, "intracellular region" describes a region of a polypeptide that is present on the intracellular (or "cytoplasmic") side of a cell surface when the polypeptide is expressed on the surface of a cell. In some embodiments, the intracellular region does not comprise a signaling domain (i.e., unable to transmit or transducing an intracellular signal). In some embodiments, the intracellular region is not derived from BCMA. In some embodiments, the intracellular region is derived from BCMA. In some embodiments, the intracellular region is derived from CD317. In some embodiments, the intracellular region is derived from CD3γ. In some embodiments, the intracellular region comprises or comprises up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, or any derivable range therein. In some embodiments, the intracellular region comprises the sequence RLR (SEQ ID NO:29). In some embodiments, the intracellular region consists of the sequence RLR (SEQ ID NO: 29). In some embodiments, the intracellular region comprises the sequence LYCWVR (SEQ ID NO: 30). In some embodiments, the intracellular region consists of the sequence LYCWVR (SEQ ID NO: 30). In some embodiments, it is identical, at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) to SEQ ID NO: 31. In some embodiments, the intracellular region comprises SEQ ID NO:31.In some embodiments, it is identical, at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) to SEQ ID NO: 32. In some embodiments, the intracellular region comprises SEQ ID NO:32. In some embodiments, it is identical, at least identical, or up to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) to SEQ ID NO: 33. In some embodiments, the intracellular region comprises SEQ ID NO:33.

[0074] In some embodiments, the chimeric polypeptide of the present disclosure comprises a signal peptide. In some embodiments, the signal peptide is a tissue-type plasminogen activator (tPA) signal peptide. In some embodiments, the signal peptide is identical, up to, or at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein) identical to SEQ ID NO:34. In some embodiments, the signal peptide comprises SEQ ID NO:34. In some embodiments, the signal peptide is CD8α signal peptide. In some embodiments, the signal peptide is identical, up to, or at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein) identical to SEQ ID NO: 35. In some embodiments, the signal peptide comprises SEQ ID NO: 35. In some embodiments, the signal peptide is GM-CSFRα signal peptide. In some embodiments, the signal peptide is identical, up to, or at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein) identical to SEQ ID NO:36.In some embodiments, the signal peptide comprises SEQ ID NO:36.

[0075] B. Sequence The amino acid sequences of certain polypeptides, including the chimeric polypeptides and portions, regions and domains thereof, are provided in Table 1.

[0076] [Table 1] JPEG2024518103000002.jpg255170JPEG2024518103000003.jpg255170JPEG20245181030 00004.jpg252170JPEG2024518103000005.jpg254170JPEG2024518103000006.jpg185170

[0077] C. Mutant Polypeptides The following is a discussion of altering the amino acid subunits of proteins to produce equivalent or even improved second generation mutant polypeptides or peptides. For example, certain amino acids can be substituted with other amino acids in a protein or polypeptide sequence with or without any appreciable loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. Because it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence and still produce a protein with similar or desirable properties. Thus, the inventors contemplate that various changes can be made to the DNA sequence of a gene encoding a protein without appreciable loss of their biological utility or activity.

[0078] The term "functionally equivalent codons" is used herein to refer to codons that code for the same amino acid, e.g., the six different codons for arginine. "Neutral substitutions" or "neutral mutations," which refer to changes in one or more codons that code for biologically equivalent amino acids, are also contemplated.

[0079] The amino acid sequence variants of the disclosure can be substitution, insertion, or deletion variants. Mutations in the polypeptides of the disclosure can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of a protein or polypeptide compared to the wild type. Variants can include at least 50%, 60%, 70%, 80%, or 90% (including all values ​​and ranges therebetween) of an amino acid sequence identical to any of the sequences provided or referenced herein. The variant may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0080] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, but may still be essentially identical as shown in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including maintenance of the biological protein activity to which protein expression pertains. The addition of terminal sequences applies in particular to nucleic acid sequences, which may contain a variety of non-coding sequences adjacent, for example, to either the 5' or 3' portion of the coding region.

[0081] Deletion mutants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted, or several consecutive amino acids can be deleted. Stop codons can be introduced (by substitution or insertion) into the encoding nucleic acid sequence to generate truncated proteins.

[0082] Insertional mutants typically include the addition of an amino acid residue at a non-terminal point of the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be made, including fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0083] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, i.e., one amino acid is replaced with one of similar chemical properties. A "conservative amino acid substitution" may involve the exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art, and include, for example, alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartate to glutamate, cysteine ​​to serine, glutamine to asparagine, glutamate to aspartate, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, valine to isoleucine or leucine. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0084] Alternatively, substitutions can be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing an amino acid residue with a chemically different one, e.g., a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitutions can involve exchanging a member of one amino acid class for a member of another class.

[0085] D. Substitution Considerations Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. Those skilled in the art can identify suitable regions of the molecule that can be changed without destroying activity by targeting regions that are not believed to be important for activity. Those skilled in the art will also be able to identify amino acid residues and portions of molecules that are conserved between similar proteins or polypeptides. In further embodiments, regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without significantly changing the biological activity or adversely affecting the protein or polypeptide structure.

[0086] In making such changes, the hydropathic index of amino acids may be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​iteratively along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al. J. Mol. Biol. 157:105-131 (1982)). It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein or polypeptide, which in turn determines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that certain amino acids may be substituted with other amino acids having similar hydropathic indexes or scores and still retain similar biological activity. In making changes based on hydropathic index, certain embodiments include substitution of amino acids whose hydropathic index is within ±2. In some aspects of the disclosure, within ±1 is included, and in other aspects of the disclosure, within ±0.5 is included.

[0087] It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigen binding, i.e., the biological properties of the protein. These amino acid residues have been assigned the following hydrophilicity values: Arginine (+3.0); Lysine (+3.0); Aspartate (+3.0±1); Glutamate (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5±1); Alanine (-0.5); Histidine (-0.5); Cysteine ​​(-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); and Tryptophan (-3.4). In making modifications based on similar hydrophilicity values, in certain embodiments, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are included, in other embodiments, substitutions of amino acids whose values ​​are within ±1 are included, and in still other embodiments, substitutions of amino acids whose values ​​are within ±0.5 are included. In some instances, epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also called "epitope core regions." It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0088] Moreover, one skilled in the art can review structure-function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in the protein that correspond to amino acid residues that are important for the activity or structure of the similar protein. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0089] The skilled artisan can also analyze the three-dimensional structure and amino acid sequence relative to that structure in similar proteins or polypeptides. In view of such information, the skilled artisan can predict the alignment of the amino acid residues of the antibody to its three-dimensional structure. The skilled artisan can choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. In addition, the skilled artisan can create test mutants that contain a single amino acid substitution at each desired amino acid residue. These mutants can then be screened using standard assays for binding and / or activity, thus obtaining information gathered from such routine experiments that may enable the skilled artisan to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the World Wide Web, for example at expasy.org / proteomics / protein_structure.

[0090] In some embodiments of the present disclosure, amino acid substitutions are made to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity for forming protein complexes, (4) change ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in portions of antibodies outside of the domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural features of the protein or polypeptide (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes a natural antibody).

[0091] EB cell maturation antigen (BCMA) B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 or TNFRSF17, is preferentially expressed on plasma cells and is highly expressed in a variety of hematological malignancies, including multiple myeloma. BCMA is encoded by the TNFRSF17 gene. An exemplary mRNA is characterized by RefSeq Accession No. NM_001192. An exemplary protein is characterized by RefSeq Accession No. NP_001183. The complete polypeptide sequence of human BCMA is provided as SEQ ID NO: 17. The complete DNA sequence of the BCMA gene (TNFRSF17) is provided as SEQ ID NO: 18.

[0092] F. Programmed cell death 1 ligand 1 (PDL1) Programmed cell death 1 ligand 1 (PDL1), also known as CD274 or B7-H1, is an immune inhibitory receptor ligand expressed by hematopoietic cells, immune cells, and various types of tumor cells. Exemplary mRNAs are characterized by RefSeq accession numbers NM_001267706 and NM_014143. Exemplary proteins are characterized by RefSeq accession numbers NP_001254635 and NP_054862.

[0093] G.CD30 CD30 is also known as tumor necrosis factor receptor superfamily member 8 or TNFRSF8 and is encoded by the TNFRSF8 gene. An exemplary mRNA is characterized by RefSeq accession number NM_001243. An exemplary protein is characterized by RefSeq accession number NP_001234.

[0094] H.Her2 Her2 (or "HER2"), also known as receptor tyrosine-protein kinase erbB-2 (erbB-2 or ErbB2), Neu or Her2 / neu, is a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases, and amplification and / or overexpression of Her2 has been reported in a number of cancers, including breast and ovarian tumors. The extracellular domain of HER2 contains four domains, domain I (about 1-195 amino acid residues), domain II (about 196-319 amino acid residues), domain III (about 320-488 amino acid residues), and domain IV (about 489-630 amino acid residues) (residue numbering without the signal peptide). See Garrett et al., Mol. Cell. 11:495-505 (2003); Cho et al., Nature 421:756-760 (2003); Franklin et al., Cancer Cell 5:317-328 (2004); Plowman et al., Proc. Natl. Acad. Sci. 90:1746-1750 (1993); and U.S. Patent No. 8,652,474, each of which is incorporated herein by reference. Her2 is encoded by the ERBB2 gene. An exemplary mRNA is characterized by RefSeq Accession No. NM_001005862. An exemplary protein is characterized by RefSeq Accession No. NP_001005862.

[0095] I.Trop2 Trop2 (or "Trop-2"), also known as tumor-associated calcium signaling agent 2, is a carcinoma-associated antigen. Trop2 is encoded by the TACSTD2 gene. An exemplary mRNA is characterized by RefSeq accession number NM_002353. An exemplary protein is characterized by RefSeq accession number NP_002344. The complete polypeptide sequence of human Trop2 is provided as SEQ ID NO: 13. The complete DNA sequence of the Trop2 gene (TACSTD2) is provided as SEQ ID NO: 14.

[0096] J. EGFR Epidermal growth factor receptor (EGFR), also known as erbB-1, is preferentially expressed on plasma cells and is highly expressed in various hematological malignancies, including multiple myeloma. The extracellular domain of EGFR is composed of four domains, which are called domain I, domain II, domain III, and domain IV, respectively, or from the N-terminus, also called L1, S1, L2, and S2 domains (see Bajaj, M. et al. Biochim. Biophys. Acta 916, 220-226 (1987) and U.S. Pat. No. 7,514,240, each of which is incorporated herein by reference). EGFR is encoded by the EGFR gene. An exemplary mRNA is characterized by RefSeq Accession No. NM_005228. An exemplary protein is characterized by RefSeq Accession No. NP_005219.

[0097] II. Nucleic acids In certain embodiments, the nucleic acid sequence may be present in various instances, such as an isolated segment of an integrated sequence or recombinant polynucleotide and recombinant vector encoding one or both chains of an antibody or a fragment, derivative, mutein, or variant thereof, a polynucleotide sufficient for use as a hybridization probe, a PCR or sequencing primer for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting expression of a polynucleotide, and a complementary sequence to the foregoing described herein. Nucleic acids encoding epitopes for which certain antibodies provided herein are provided are also provided. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids may be single-stranded or double-stranded and may comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0098] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. Polynucleotides, in certain embodiments, include regulatory sequences that are isolated substantially away from their naturally occurring gene or protein coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present within the polynucleotide.

[0099] In this regard, the terms "gene", "polynucleotide" or "nucleic acid" are used to refer to a nucleic acid that encodes a protein, polypeptide or peptide (including any sequences necessary for proper transcription, post-translational modification, or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide may contain a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid including variants having slightly different nucleic acid sequences, but that nevertheless encode the same or substantially similar proteins.

[0100] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein, including at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis with standard parameters). In certain aspects, the isolated polynucleotide, or a nucleotide sequence complementary to the isolated polynucleotide, will comprise a nucleotide sequence that encodes a polypeptide having at least 90%, preferably 95% or more identity over the entire length of the sequence, to the amino acid sequence described herein.

[0101] Nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and their total length may vary considerably. Nucleic acids may be of any length. They may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or may include one or more additional sequences, such as regulatory sequences, and / or may be part of a larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of almost any length may be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic benefits such as targeting or efficacy. As noted above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, with "heterologous" referring to a polypeptide that is not the same as the modified polypeptide.

[0102] A. Hybridization A nucleic acid that hybridizes to another nucleic acid under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide hybridization buffer, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing about 50% formamide and having a hybridization temperature of 42°C), and washing conditions of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions include hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the stringency of hybridization, such that nucleic acids comprising nucleotide sequences that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other will typically remain hybridized to each other.

[0103] Parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are provided, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated by reference in their entireties for all purposes, and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0104] B. Mutation Changes can be introduced by mutations into the nucleic acid, thereby resulting in a change in the amino acid sequence of the polypeptide (e.g., an antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, for example, using a random mutagenesis protocol. However, once created, the mutant polypeptide can be expressed and screened for desired properties.

[0105] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively alters the biological activity of the polypeptide it encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, decreasing or eliminating activity. Examples of qualitative changes include changing the antigen specificity of an antibody.

[0106] III. Infinite immune cells Certain embodiments of the present disclosure relate to immune cells engineered to express one or more genes. The expression of one or more genes directly or indirectly results in increased life span of the cell compared to a cell lacking expression of one or more genes. In certain embodiments, the cell is engineered to express one or more genes, including one or more heterologous genes. In other cases, the cell is engineered to have upregulation of expression of one or more genes that are endogenous to the cell, such as by manipulation of one or more regulatory elements of one or more endogenous genes to the cell.

[0107] In certain embodiments, immune cells are engineered to express BCL6 and one or more pro-survival or anti-apoptotic or cell survival genes (and there may or may not be overlap in genes classified as pro-survival or anti-apoptotic or cell survival). As used herein, pro-survival genes refer to nucleic acid polymers that can exert anti-apoptotic functions or promote survival by any mechanism. Nucleic acid polymers that can exert anti-apoptotic functions can be one or more of the Bcl2 family genes, such as BCL-xL, BCL-2, MCL-1, Bcl-w, Bfl-1, BCL-B. Nucleic acid polymers that can exert anti-apoptotic functions can be one or more of the inhibitor of apoptosis (IAP) family genes, such as XIAP, c-IAP1, C-IAP2, NAIP, and survivin. The nucleic acid polymer that can exert an anti-apoptotic function may be capable of inhibiting or knocking out the expression of one or more caspases that play a role in apoptosis, such as caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14. The nucleic acid polymer for knockdown or knockout may be an shRNA expression cassette, or these caspase genes may also be knocked out by gene editing methods (CRISPR, TALEN, zinc finger method, etc.). The nucleic acid polymer that can exert an anti-apoptotic function may be capable of inhibiting or knocking out the expression of one or more pro-apoptotic genes, such as BIM, Puma, Noxa, Bik, Bmf, Bad, Hrk, Bid, BAX, BAK, BOK, etc.Nucleic acid polymers capable of exerting an anti-apoptotic function may have the anti-apoptotic effect of insulin-like growth factor (IGF-1), Hsp70, Hsp27, cFLIP, BNIP3, FADD, Akt and NF-κB, Raf-1 and MEK1, p90Rsk, C-Jun, BNIP2, BAG1, HSPA9, HSP90B1, miRNA21, miR-106b-25, miR-206, miR-221 / 222, miR-17-92, miR-133, miR-143, miR-145, miR-155, miR-330, and the like.

[0108] Infinite T cells can be made with either wild-type or mutant BCL6. The inventors have determined that infinite T cells can be made with either wild-type or mutant BCL6 with a single specific nucleotide difference - the codon for the amino acid at position 395 in wild-type BCL6 is CCT (encoding proline / P) and the codon for the amino acid at position 395 in mutant BCL6 is CTT (encoding leucine / L). The nucleotide and amino acid sequences of the two BCL6 genes are shown below (the mutation point in the wild-type sequence is underlined):

[0109] Wild type BCL6 aa sequence:

[0110] (SEQ ID NO:43)

[0111] Nucleotide sequence of wild-type BCL6 (mutation codons in the wild-type sequence are underlined):

[0112] cCt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactctacatgcaccccccgaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagacccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgctgccaggcctccttccgctacaagggcaacctcgccagccacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaaccggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttccggcaccttcagactctgaagagccacctgcgaatccacacaggagagaaaccttaccattgtgagaagtgtaacctgcatttccgtcacaaaagccagctgcgacttcacttgcgccagaagcatggcgccatcaccaacaccaaggtgcaataccgcgtgtcagccactgacctgcctccggagctccccaaagcctgc(SEQ ID NO: 44)

[0113] Mutant BCL6 aa sequence (leucine mutations are underlined):

[0114] MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEG L EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC (SEQ ID NO: 45)

[0115] Nucleotide sequence of mutant BCL6 (leucine codons are underlined):

[0116] cTt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactctacatgcaccccccgaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagacccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgctgccaggcctccttccgctacaagggcaacctcgccagccacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaaccggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttccggcaccttcagactctgaagagccacctgcgaatccacacaggagagaaaccttaccattgtgagaagtgtaacctgcatttccgtcacaaaagccagctgcgacttcacttgcgccagaagcatggcgccatcaccaacaccaaggtgcaataccgcgtgtcagccactgacctgcctccggagctccccaaagcctgc (SEQ ID NO: 46)

[0117] Immune cells include T cells (e.g. regulatory T cells, CD4 + T cells, CD8 + The immune cells may be any type of immune cell, including T cells, alpha beta T cells, gamma-delta T cells, or mixtures thereof), NK cells, invariant NKT cells, NKT cells, innate lymphoid cells, or mixtures thereof. The immune cells may be virus-specific, may express a CAR, and / or may express a TCR. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. Also provided herein are methods of producing and manipulating immune cells, as well as methods of using and administering cells for adoptive cell therapy, where the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, for example to target cancer cells. These immune cells may be used therapeutically as a single cell type or as a combination of multiple immune cell types. In certain embodiments, the immune cells are CD3+, CD4+, CD8+, CD16+, or mixtures thereof.

[0118] Immune cells can be isolated from a subject, particularly a human subject. Immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject undergoing treatment for a particular disease or condition. Immune cells can be collected from any location present in a subject, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used directly or can be stored for a period of time, such as by freezing.

[0119] Immune cells may be enriched / purified from any tissue in which they reside, including, but not limited to, blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. The tissues / organs from which immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, where the non-living subject is an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some embodiments, immune cells isolated from umbilical cord blood have enhanced immunomodulatory capabilities, as measured by CD4 or CD8 positive T cell suppression. In certain embodiments, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, to enhance immunomodulatory capabilities. Pooled blood may be from two or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0120] The population of immune cells can be obtained from a subject who needs treatment or suffers from a disease associated with reduced immune cell activity. Thus, the cells are autologous to the subject who needs treatment. Alternatively, the population of immune cells can be obtained from a donor, such as a partially or fully histocompatible donor or a fully histocompatible donor. The immune cell population can be taken from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood.

[0121] When the population of immune cells is obtained from a donor different from the subject, the donor can be allogeneic, so long as the obtained cells are subject-compatible in that they can be introduced into the subject. Allogeneic donor cells may or may not be human-leukocyte-antigen (HLA) compatible.

[0122] Further methods and compositions related to Infinite Immune Cells are described in PCT Patent Application Publication No. WO / 2021 / 034982, which is incorporated by reference in its entirety.

[0123] AT cells In some embodiments, the immune cells are T cells. Several basic approaches for the induction, activation and expansion of functional anti-tumor effector cells have been described in the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs), autologous DCs or PBMCs, lymphocytes, artificial antigen-presenting cells (APCs), or T cells activated ex vivo using beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes, allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs), and non-tumor specific autologous or allogeneic cells that have been genetically reprogrammed or "retargeted" to express tumor-reactive TCRs or chimeric TCR molecules that exhibit antibody-like tumor recognition capabilities known as "T-bodies". These approaches have given rise to a number of protocols for T cell preparation and immunization that can be used in the methods described herein.

[0124] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as those isolated directly from a subject and / or those isolated from a subject and frozen. In some embodiments, the cells are one or more subsets of T cells or other cell types, such as the total T cell population, CD4 + cells, CD8 +The present invention includes cells and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, proliferation, recirculation, localization, and / or persistence ability, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. The cells can be allogeneic and / or autologous with respect to the subject being treated. In some aspects, such as off-the-shelf technology, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating the cells from the subject, preparing, treating, culturing and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0125] T cells (e.g., CD4 + and / or CD8 + Among the subtypes and subpopulations of T cells, there are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ), Central Memory T (TC M ), Effector Memory T (T EM ), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and gamma / delta T cells.

[0126] In some embodiments, one or more T cell populations are positive for a particular marker, such as a surface marker, or are enriched or depleted in cells that are negative for a particular marker. In some cases, such markers are markers that are absent or expressed at relatively low levels in certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels in certain other populations of T cells (e.g., memory cells).

[0127] In some embodiments, T cells are isolated from the PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD14. + or CD8 + Using a selection process, CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated from CD4 + and CD8 + The population can be further divided into subpopulations by positive or negative selection for markers that are expressed or relatively highly expressed on one or more naive, memory and / or effector T cell subpopulations.

[0128] In some embodiments, CD8 + The T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM ) cells or stem cell memory cells are enriched to enhance efficacy, e.g., to improve long-term survival, proliferation, and / or engraftment following administration, which in some embodiments is particularly robust in such subpopulations.

[0129] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension can be obtained by any suitable method, for example mechanically (e.g., disrupting the tumor using a GENTLEMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). The single cell suspension of the tumor enzymatic digest is cultured in interleukin-2 (IL-2) or other growth factors.

[0130] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides at least about a 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) increase in the number of antigen-specific T cells over a period of about 10 to about 14 days. More preferably, rapid expansion provides at least about a 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or more) increase over a period of about 10 to about 14 days.

[0131] Expansion can be achieved by any of a number of methods known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Non-specific T cell receptor stimulation can include about 30 ng / ml OKT3, a murine monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, NJ). Alternatively, T cells can be rapidly expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more antigens of the cancer (including antigenic portions thereof, such as epitopes, or cells), which may optionally be expressed from a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides or peptides that bind to other MHC class I or class II molecules, in the presence of a T cell growth factor, such as 300 IU / ml IL-2 or IL-15, with IL-2 being preferred. In vitro induced T cells rapidly proliferate upon restimulation with the same antigen of the cancer pulsed onto HLA-A2 expressing antigen presenting cells or antigen presenting cells expressing other HLA molecules. In vitro induced T cells can also be proliferated in the absence of antigen presenting cells.

[0132] Autologous T cells can be modified to express T cell growth or differentiation factors that promote the growth, differentiation, and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, IL-18, IL-21, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001 and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley&Sons, NY, 1994. In certain embodiments, the modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences, such as those for IL-12, are readily available in the art, as are promoters whose operable linkage to the T cell growth factor coding sequences promotes high levels of expression.

[0133] B.NK cells In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in bone marrow and thymus. NK cells differentiate and mature in bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers such as CD16, CD56, and / or CD8 in humans. NK cells do not express T cell antigen receptor, panT marker CD3, or surface immunoglobulin B cell receptor.

[0134] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissue, or umbilical cord blood by methods well known in the art.

[0135] C.NKT cells Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only about 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express the αβ T cell receptor but also express various molecular markers typically associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells express high levels and depend on the transcriptional regulator promyelocytic leukemia zinc finger for their development. Currently, there are five major distinct iNKT cell subsets. These subset cells produce distinct sets of cytokines upon activation. The subtypes iNKT1, iNKT2, and iNKT17 mirror Th cell subsets in cytokine production. In addition, there are subtypes specialized for follicular helper-like T function and IL-10-dependent regulatory function.

[0136] D. Innate lymphocytes Innate lymphoid cells (ILCs) are a group of innate immune cells that originate from common lymphoid progenitors (CLPs) and belong to the lymphoid lineage. These cells are defined by the absence of antigen-specific B or T cell receptors due to the lack of recombination activation genes (RAGs). ILCs do not express myeloid or dendritic cell markers. Since they play a role in the regulation of protective immunity as well as homeostasis and inflammation, their dysregulation can result in immunopathologies such as allergy, bronchial asthma and autoimmune diseases. ILCs are divided based on the cytokines they can produce, as well as the transcription factors that regulate their development and function.

[0137] IV. Cell Preparation and Culture In certain embodiments, the cells of the present disclosure may be specifically formulated and / or cultured in a particular medium. The cells may be formulated so as to be suitable for delivery to a recipient without adverse effects.

[0138] In a certain embodiment, the medium may be prepared using any of media used for culturing animal cells, such as AIMV, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, GlasgowMEM, ImprovedMEMZincOption, IMDM, Medium199, EagleMEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer medium, and any combination thereof, as the basal medium, but the medium is not particularly limited thereto, as long as it can be used for culturing animal cells. In particular, the medium may be xeno-free or chemically defined.

[0139] The medium may be a serum-containing or serum-free medium, or a xeno-free medium. In order to prevent contamination with components derived from different animals, the serum may be derived from the same animal as the stem cells. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium that contains purified blood-derived components or animal tissue-derived components (such as growth factors).

[0140] The medium may or may not contain a serum replacement. Serum replacements may include materials that suitably contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant or humanized albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum replacements can be prepared, for example, by the methods disclosed in WO 98 / 30679, which is incorporated herein in its entirety. Alternatively, for greater convenience, any commercially available material can be used. Commercially available materials include KNOCKOUT™ Serum Replacement (KSR), Chemically-Defined Lipid Concentrate (GIBCO™), GLUTAMAX™ (GIBCO™).

[0141] In certain embodiments, the medium may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins such as biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A (acetate), proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free fraction V, catalase, human recombinant insulin, human transferrin, superoxide dismutase, other components such as corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and / or T3 (triiodo-I-thyronine). In certain embodiments, one or more of these may be explicitly excluded.

[0142] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any derivable range therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of B-27® supplement, XenoFree B-27™ supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, simple sugars, inorganic ions.In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In some embodiments, the medium further includes one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-I-thyronine, B-27™ supplement, XenoFree B-27™ supplement, GS21™ supplement, amino acids (e.g., arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus, etc.) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain embodiments, one or more of these may be explicitly excluded.

[0143] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, in certain embodiments, one or more of which may be explicitly excluded.

[0144] One or more of the media components may be added at a concentration of at least, up to, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any derivable range therein.

[0145] In certain embodiments, the cells of the present disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, with certain formulations including 2.5% human albumin. The cells may be specifically formulated for intravenous administration. For example, they are formulated for intravenous administration over less than one hour. In certain embodiments, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from the time of thawing.

[0146] In some embodiments, the cells of the present disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which a CAR can be directed include at least 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvI, and / or EGFRvIII. II, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), Her2, IL-13Rα2, lambda, Lewis Y, kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, Examples of such proteins include PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPVE6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, such as Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). A CAR can be a first, second, third or higher generation CAR. A CAR can be bispecific for any two non-identical antigens, or specific for three or more non-identical antigens.

[0147] In some embodiments, the cells of the present disclosure comprise one or more chimeric polypeptides. The cells may comprise a chimeric polypeptide of the present disclosure together with a chimeric antigen receptor, a T cell receptor, and / or other engineered receptor or molecule. In some embodiments, the cells of the present disclosure comprise one, two, three, four, five or more chimeric polypeptides.

[0148] V. Chimeric Antigen Receptor Certain embodiments of the present disclosure relate to chimeric antigen receptors (CARs), cells comprising one or more CARs, and methods of their use.

[0149] A. Signal Peptides The polypeptides of the present disclosure may include a signal peptide. "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example to a certain cell organelle (such as the endoplasmic reticulum) and / or to the cell surface. In some embodiments, the signal peptide directs the nascent protein to the endoplasmic reticulum, which is essential when the receptor is glycosylated and anchored to the cell membrane. In general, the signal peptide that is naturally associated with the most amino-terminal component is used (e.g., in an scFv with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0150] In some embodiments, the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide, hi some embodiments, a restriction site is at the carboxy terminus of the signal peptide to facilitate cleavage.

[0151] B. Antigen-binding domain The polypeptides of the present disclosure may comprise one or more antigen-binding domains. An "antigen-binding domain" refers to a region of a polypeptide that can bind to an antigen under appropriate conditions. In some embodiments, the antigen-binding domain is one or more antibody-based single-chain variable fragments (scFvs). In some embodiments, the antigen-binding domain comprises a variable heavy (VH) region and a variable light (VL) region, where the VH region and the VL region are on the same polypeptide. In some embodiments, the antigen-binding domain comprises a linker between the VH region and the VL region. The linker may enable the antigen-binding domain to form a desired structure for antigen binding.

[0152] The variable regions of the antigen-binding domain of the polypeptide of the present disclosure can be modified by mutating amino acid residues in the VH and / or VL CDR1, CDR2 and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to the complementarity determining region based on a portion of the variable chains of immunoglobulins (antibodies) and T cell receptors, which are produced by B cells and T cells, respectively, and which bind to their specific antigens. Since most sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties of interest can be evaluated in suitable in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically altering no more than 1, 2, 3, 4 or 5 residues in the CDR regions. Mutations can be amino acid substitutions, additions or deletions.

[0153] Framework modifications can be made to an antibody to reduce immunogenicity, e.g., by "backmutating" one or more framework residues to the corresponding germline sequence.

[0154] It is also contemplated that an antigen-binding domain may be made multispecific or multivalent by multimerizing the antigen-binding domain with a VH domain pair and a VL domain pair that bind either the same antigen (multivalent) or different antigens (multispecific).

[0155] The binding affinity of an antigen-binding region, such as a variable region (heavy and / or light chain variable region), or a CDR, is at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 In some embodiments, the KD of an antigen-binding region, such as a variable region (heavy and / or light chain variable region), or the KD of a CDR, can be at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M (or any derivable range therein).

[0156] The binding affinity, K, or KD can be determined by methods known in the art, such as surface plasmon resonance (SRP)-based biosensors, kinetic exclusion assays (KinExA), optical scanners for microarray detection based on polarization-modulated grazing incidence reflectance difference (OI-RD), or ELISA.

[0157] In some embodiments, a polypeptide comprising a humanized binding region has equal, better, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115, or 120% binding affinity and / or expression level in a host cell compared to a polypeptide comprising a non-humanized binding region, such as a binding region from a mouse. In some embodiments, framework regions such as FR1, FR2, FR3 and / or FR4 of the human framework, individually or collectively, are at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 1 The sequence may have 63, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions.

[0158] In some embodiments, the framework regions such as FR1, FR2, FR3 and / or FR4 of the mouse framework, individually or collectively, are at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 110 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 1 The sequence may have 63, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions.

[0159] The substitutions are at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, It may be in position 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0160] C. Peptide Spacer A peptide spacer, such as an extracellular spacer, may link the antigen-binding domain to the transmembrane domain. In some embodiments, the peptide spacer is sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen binding. In one embodiment, the spacer comprises a hinge region derived from IgG. In some embodiments, the spacer comprises or further comprises a CH2CH3 region and a portion of CD3 of an immunoglobulin. In some embodiments, the CH2CH3 region may have a L235E / N297Q or L235D / N297Q modification, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to the CH2CH3 region. In some embodiments, the spacer is derived from IgG4. The extracellular spacer may comprise a hinge region.

[0161] As used herein, the term "hinge" refers to a flexible polypeptide connector region (also referred to herein as "hinge region") that provides structural flexibility and spacing to adjacent polypeptide regions and may be composed of natural or synthetic polypeptides. A "hinge" derived from an immunoglobulin (e.g., IgG1) is generally defined as the extension from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions. Hinge regions may be naturally occurring or non-naturally occurring, including but not limited to modified hinge regions as described in U.S. Pat. No. 5,677,425, which is incorporated herein by reference. Hinge regions may include a complete hinge region derived from an antibody of a class or subclass different from that of the CH1 domain. The term "hinge" can also include regions derived from CD8 and other receptors that serve a similar function in providing flexibility and spacing to adjacent regions.

[0162] The extracellular spacer may be at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 27 It can have a length of 16, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any derivable range therein). In some embodiments, the extracellular spacer consists of or comprises a hinge region from an immunoglobulin (e.g., IgG). Immunoglobulin hinge region amino acid sequences are known in the art. See, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res.

[0163] The length of the extracellular spacer can affect the signaling activity of the CAR in response to antigen-stimulated CAR signaling and / or the proliferation properties of the CAR-T cells. In some embodiments, shorter spacers are used, such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids. In some embodiments, longer spacers are used, such as at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 222, 224, 226, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, Those that are 5, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280 or 290 amino acids may have the advantage of increased growth in vivo or in vitro.

[0164] When the extracellular spacer comprises multiple moieties, there can be anywhere from 0 to 50 amino acids between the various moieties. For example, there can be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and CH2 or CH3 regions, or if both are present, between the CH2 and CH3 regions. In some embodiments, the extracellular spacer consists essentially of the hinge, CH2, and / or CH3 regions, meaning that the hinge, CH2, and / or CH3 regions are the only identifiable regions present, and all other domains or regions are excluded, although there may be additional amino acids that are not part of the identifiable regions.

[0165] D. Transmembrane domain The polypeptides of the present disclosure may include a transmembrane domain. In some embodiments, the transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may confer different receptor stabilities.

[0166] In some embodiments, the transmembrane domain is sandwiched between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is sandwiched between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is between the transmembrane domain and one or more costimulatory regions.

[0167] Any transmembrane domain that provides for insertion of the polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD28, CD8, CD4, CD3-zeta (CD3ζ), CD134, or CD7.

[0168] E. Cytoplasmic region After antigen recognition, the receptors of the present disclosure cluster and a signal can be transmitted to the cell through the cytoplasmic region. In some embodiments, the costimulatory domain described herein is part of the cytoplasmic region. In some embodiments, the cytoplasmic region comprises an intracellular signaling domain. The intracellular signaling domain can comprise a primary signaling domain and one or more costimulatory domains.

[0169] Cytoplasmic and / or costimulatory regions suitable for use in the polypeptides of the present disclosure include any desired signaling domain that provides a distinct detectable signal (e.g., increased production of one or more cytokines by the cell, changes in transcription of a target gene, changes in protein activity, changes in cellular behavior such as cell death, cell proliferation, cell differentiation, cell survival, modulation of a cell signaling response, etc.) in response to activation by binding of an antigen to the antigen-binding domain. In some embodiments, the cytoplasmic region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region comprises a DAP10 / CD28-type signaling chain.

[0170] Cytoplasmic regions suitable for use in the polypeptides of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region includes 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the endodomain, and the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids, e.g., (YX1X2(L / I)(X3)n(YX1X2(L / I), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid).

[0171] A suitable cytoplasmic region can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable endodomain does not need to include the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0172] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that can be incorporated into the CARs of the present disclosure.

[0173] In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence.

[0174] In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3ε; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3-epsilon, T3e, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3γ, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3γ, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence.

[0175] In some embodiments, the cytoplasmic region is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein; etc.). In some embodiments, a suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD79A amino acid sequence.

[0176] F. Co-stimulatory area Non-limiting examples of suitable costimulatory regions, such as those contained in the cytoplasmic region, include, but are not limited to, polypeptides derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0177] The costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any derivable range therein. In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the costimulatory region is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0178] G. Detection Peptide In some embodiments, the polypeptides described herein may further comprise a detection peptide. A variety of suitable detection peptides are known in the art and are contemplated herein.

[0179] H. Peptide Linker In some embodiments, the polypeptides of the present disclosure include a peptide linker (sometimes referred to as a linker). Peptide linkers can be used to separate any of the peptide domains / regions described herein. By way of example, a linker can be present between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, adjacent to the costimulatory region or on the N- or C-region of the costimulatory region, and / or between the transmembrane domain and the endodomain. The peptide linker can have any of a variety of amino acid sequences. The domains and regions can be linked by peptide linkers, which are generally flexible in nature, although other chemical bonds are not excluded. The linker can be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length. These linkers can be produced by linking proteins using oligonucleotides encoding synthetic linkers.

[0180] Peptide linkers with some degree of flexibility can be used. The peptide linker can have virtually any amino acid sequence, keeping in mind that suitable peptide linkers generally have sequences that result in flexible peptides. The use of small amino acids such as glycine and alanine is useful for creating flexible peptides. The creation of such sequences is routine for those skilled in the art.

[0181] A suitable linker can be readily selected and can be of any suitable length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0182] Suitable linkers can be readily selected and can be of any of a variety of lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0183] VI.Cells Certain embodiments relate to cells comprising the polypeptide or nucleic acid of the present disclosure. In some embodiments, the cell is an immune cell or a T cell. "T cell" includes all types of immune cells that express CD3, including T helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, regulatory T cells (Treg) gamma-delta T cells, natural killer (NK) cells, and neutrophils. T cells may refer to CD4+ or CD8+ T cells.

[0184] Suitable mammalian cells include primary cells and immortalized cell lines, including human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0185] In some cases, the cells are not immortalized cell lines, but rather are cells (e.g., primary cells) obtained from an individual. For example, in some cases, the cells are immune cells obtained from an individual. As one example, the cells are T lymphocytes obtained from an individual. As another example, the cells are cytotoxic cells obtained from an individual. As another example, the cells are stem cells (e.g., peripheral blood stem cells) or progenitor cells obtained from an individual.

[0186] VII. Cytokines In some embodiments, any cell encompassed herein may express one or more heterologous cytokines. In certain embodiments, the cells are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with one or more cytokines on the same vector as other genes. In some cases, in addition to one or more cytokines, the cells also express one or more heterologous proteins, including any type of chimeric protein. As an example, the cells may express a chimeric antigen receptor.

[0187] One or more cytokines may be co-expressed from the vector as a polypeptide separate from other proteins, including the chimeric polypeptide and / or CAR. For example, interleukin-15 (IL-15) is tissue restricted and is only observed at any level in serum or systemically under pathological conditions. IL-15 has several desirable attributes for adoptive therapy. IL-15 is a homeostatic cytokine that induces natural killer cell development and cell proliferation, promotes eradication of established tumors via relief of functional inhibition of tumor-resident cells, and inhibits activation-induced cell death (AICD). In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. NK cells expressing IL-15 are capable of sustained supportive cytokine signaling, which is useful for survival after infusion.

[0188] In certain embodiments, the cells express one or more exogenously provided cytokines. By way of example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. Additionally or alternatively, the cytokines can be exogenously provided to the cells. In alternative cases, endogenous cytokines in the cells are upregulated by manipulating the regulation of expression of the endogenous cytokine, such as by genetic modification at the promoter site of the cytokine. When the cytokine is provided to the cells on an expression construct, the cytokine can be encoded from the same vector as other heterologous proteins.

[0189] In some embodiments, the heterologous protein utilized in the cell is a fusion of a cytokine with at least a portion of its receptor, including part or all of the extracellular domain of that receptor. In certain cases, part or all of IL-15 is fused to part or all of the IL-15Ra receptor, e.g., part or all of the extracellular domain of the IL-15Ra receptor, e.g., the sushi domain.

[0190] VIII. General Pharmaceutical Compositions In some embodiments, the pharmaceutical composition is administered to a subject. Different aspects may include administering an effective amount of the composition to a subject. In some embodiments, cell therapy (e.g., CART cells, CARNK cells, TCRT cells, etc.) is administered to a subject to protect against or treat a condition (e.g., cancer). Furthermore, such compositions can be administered in combination with additional therapeutic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, etc.). Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0191] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Other supplementary active ingredients, such as anti-infective agents and vaccines, can also be incorporated into the composition.

[0192] The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or intraperitoneal route.Typically, such compositions can be prepared as either liquid solutions or suspensions.Solid forms can also be prepared that are suitable for use in preparing solutions or suspensions by adding liquid before injection.The preparation can also be emulsified.

[0193] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, such as formulations with aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that it can be easily injected. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0194] Proteinaceous compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0195] The pharmaceutical compositions may contain, for example, a solvent or dispersion medium containing water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0196] Sterile injectable solutions are prepared by incorporating the required amount of active compound into a suitable solvent with various other ingredients as listed above, as necessary, followed by filtration sterilization or equivalent procedures.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredient from its previously sterile-filtered solution.

[0197] Administration of the compositions is typically via any common route, including, but not limited to, oral or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intranasal administration. Such compositions are usually administered as pharma- ceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0198] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injection solutions described above.

[0199] IX. Chimeric Polypeptides as Transduction Markers and / or Safety Switches Embodiments of the present disclosure include chimeric polypeptides that can be utilized as transduction markers and / or safety switches. Safety strategies can be utilized, for example, with cell therapy as a means to overcome toxicity if it occurs. Examples that exist in the art include herpes simplex virus thymidine kinase (HSV-tk) / ganciclovir, inducible caspase 9, and truncated EGFR gene (tEGFR). EGFR gene primarily uses antibody-dependent cellular cytotoxicity (ADCC) mechanisms to eliminate therapeutic cells, and ADCC requires the presence of NK cells and other effector cells that may limit the effectiveness of such safety switches in vivo. Ideally, the safety switch would utilize ADCC or antibody-drug conjugate (ADC) mechanisms to eliminate therapeutic cells in vivo. The ADC approach does not require effector cells for the elimination of therapeutic cells in vivo and is therefore expected to be more effective in vivo. Furthermore, tEGRF safety switches may have limited or no effectiveness in patients recently treated with, for example, lymphodepleting chemotherapy.The present disclosure provides a solution to a need in the area of ​​safety for cell therapies.

[0200] The embodiments of the present disclosure include methods and compositions related to cell therapies that have a lower risk of adverse effects compared to cell therapies that are not similarly produced. In certain embodiments, the therapeutic cells of the present disclosure express at least one protein marker that can also be used as a safety switch, for example, if the cell therapy becomes toxic to the recipient individual. In some cases, the protein is utilized as a marker rather than a safety switch, in some cases, the protein is used as a safety switch rather than a marker, and in other cases, the protein is utilized as both a marker and a safety switch. The therapeutic cells may or may not be monitored using a marker prior to the onset of any toxicity. The marker / safety switch may also be useful for monitoring the production of the cells and / or the use of the cells.

[0201] In certain embodiments, the chimeric polypeptide is used not only as a marker for a particular cell, but also as a safety switch to kill the cell if necessary. In some embodiments, the chimeric polypeptide is present in a cell where it is not normally expressed, and / or the chimeric polypeptide is present in a cell as any kind of heterologous protein, possibly as a fusion protein. In certain embodiments, a chimeric polypeptide fusion protein that includes at least the extracellular domain of a particular protein is used both as a marker for the cell that expresses it, and as a target to kill the cell that expresses it, if desired. In certain cases, the chimeric polypeptide is present as a fusion protein in which the cytoplasmic domain is not native to any part of the rest of the protein (although the transmembrane domain of the fusion protein may or may not be the transmembrane domain found in nature with the extracellular domain), and in some aspects, the cytoplasmic domain is the cytoplasmic domain of a cell receptor, such as an internalizing cytoplasmic domain. In certain cases, the cytoplasmic domain of the chimeric polypeptide fusion protein is derived from CD30, B-cell maturation antigen (BCMA), tumor-associated calcium signaling agent 2 (trop-2), CD317, CD3 gamma, CD4, CD79b, CD19, CD22, CD25, CD33, or a combination thereof.

[0202] The methods of the disclosure include methods of identifying cells that express at least a chimeric polypeptide (e.g., a polypeptide that includes at least a portion of a particular extracellular domain such that an agent that binds to the extracellular domain, such as an antibody, can bind to it). Identification of cells using a particular protein can be for any reason, such as to determine the quality of production of cells expressing it, to monitor the location of the cells, and / or to determine the quantity of the cells.

[0203] In certain embodiments, the chimeric polypeptide comprises a portion or the entirety of the CD30 extracellular domain. In certain cases, the cells of the cell therapy are controlled by targeting CD30 expressed on the cells. For example, if the recipient of the cells shows any indication that the cell therapy has become harmful in some way, such as being toxic to the individual, the control of the CD30 positive cells allows them to be inhibited, including killed. In some embodiments, the CD30 positive cells are of any type and express any type of heterologous CD30 protein, including chimeric fusion proteins. In other embodiments, the natural machinery of the cells causes the cells to express CD30 under non-native conditions. For example, CD30 can be expressed after transfection or transduction of one or more heterologous genes, including certain combinations of heterologous genes. In certain cases, transfection or transduction of BCL6 and BCL2L1 in cells results in the expression of CD30 (including cells that do not normally express CD30), and in such cases, CD30 can be used as a safety switch (or marker). In certain embodiments, if a recipient individual exhibits toxicity to a CD30 positive cell therapy, the individual is provided with an effective amount of one or more agents, e.g., an antibody or antibody-drug conjugate, that target CD30 and include a monoclonal antibody; a CD30-targeted chimeric antigen receptor-expressing immune cell (T cell (including αβ or γδ), NK cell, NKT cell, monocyte, macrophage, B cell, mesenchymal stem cell (MSC) cell, hematopoietic stem cell (HSC), hematopoietic cell, induced pluripotent stem cell (iPSC) or derivatives thereof, mixtures thereof, derivatives thereof); a CD30 / CD3 bispecific antibody; or a CD30 / CD16 bispecific antibody.

[0204] Embodiments of the present disclosure include expression constructs that include sequences encoding certain chimeric polypeptides, where the chimeric polypeptides include at least a portion of a particular extracellular domain fused to an intracellular domain that includes endocytosis or internalization activity. In certain cases, the cytoplasmic domain of the chimeric polypeptide fusion protein is derived from CD30, B-cell maturation antigen (BCMA), tumor-associated calcium signaling agent 2 (trop-2), CD317, CD3 gamma, CD4, CD79b, CD19, CD22, CD25, CD33, or combinations thereof. In certain cases, the chimeric polypeptide may or may not include a particular transmembrane domain, e.g., a transmembrane domain that is the alpha or beta chain of the T cell receptor, or a transmembrane domain from CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154. In certain embodiments, the fusion protein may comprise SEQ ID NO: 47 and the sequence encoding the fusion protein may comprise SEQ ID NO: 48. Any of the expression constructs encompassed herein may be present in a vector, including a viral vector or a non-viral vector.

[0205] Certain embodiments of the present disclosure include isolated cells comprising any expression construct encompassed herein, such as immune cells. The cells may be αβT cells, γδT cells, NK cells, NKT cells, monocytes, macrophages, B cells, mesenchymal stem cells (MSC) cells, hematopoietic stem cells (HSC), hematopoietic cells, iPSCs, or mixtures thereof. In some cases, the cells express one or more heterologous proteins other than the chimeric polypeptide. Any heterologous protein may be a therapeutic protein, a cytokine, a fusion of a cytokine with a cytokine receptor, a safety switch, or mixtures thereof. In certain cases, the therapeutic protein is an engineered antigen receptor, an antibody, or the like. The engineered antigen receptor may target a cancer antigen. The engineered antigen receptor may be a chimeric antigen receptor, a T cell receptor, or a B cell receptor. In certain embodiments of the cells, the chimeric polypeptide and the one or more heterologous proteins are expressed from the same vector, while in other cases, the chimeric polypeptide and the one or more heterologous proteins are expressed from different vectors. The cells may express heterologous BCL6 and one or more Bcl2 family genes. Embodiments of the present disclosure include an isolated population of any one of the cells encompassed herein in a suitable medium. The population may be stored in an archive and / or cryopreserved.

[0206] Certain embodiments of the present disclosure include a method of identifying CD30 positive cells transduced or transfected with (1) a heterologous CD30 gene, (2) a CD30 fusion protein comprising at least a portion of the CD30 extracellular domain, or (3) a combination of heterologous BCL6 and one or more Bcl2 family genes, comprising the steps of transducing or transfecting a cell with (1) a heterologous CD30 gene, (2) a CD30 fusion protein comprising at least a portion of the CD30 extracellular domain, or (3) a combination of heterologous BCL6 and one or more Bcl2 family genes, exposing the cell to an effective amount of an agent that binds to CD30, and directly or indirectly detecting binding of the agent to CD30 on the surface of the cell. The exposing and detecting steps may occur during and / or after production of the cells. The method may further include a step of producing the cells. In certain cases, the method is further defined as transfecting or transducing an immune cell with a CD30 fusion protein, exposing the immune cell to an effective amount of an antibody or antibody-drug conjugate that binds to CD30, and directly or indirectly detecting binding of the antibody or antibody-drug conjugate to CD30 on the surface of the cell. The method may be further defined as transfecting or transducing an immune cell with heterologous BCL6 and one or more Bcl2 family genes, exposing the immune cell to an effective amount of an antibody or antibody-drug conjugate that binds to CD30, and directly or indirectly detecting binding of the antibody or antibody-drug conjugate to CD30 on the surface of the cell. In certain cases, the method is performed in vitro, although in some cases, at least a portion of the method is performed in vivo. The cell may express one or more heterologous proteins other than the CD30 fusion protein, such as a therapeutic protein, a cytokine, a fusion of a cytokine with a cytokine receptor, a safety switch, or a mixture thereof. The therapeutic protein may be an engineered antigen receptor. In certain embodiments, the method further comprises transfecting or transforming the immune cells with a heterologous protein other than a heterologous CD30 or CD30 fusion protein, or with BCL6 and one or more Bcl2 family genes.In some embodiments, the CD30 fusion protein and a heterologous protein other than the CD30 fusion protein, or BCL6 and one or more Bcl2 family genes are expressed from the same vector. The CD30 fusion protein and a heterologous protein other than the CD30 fusion protein, or BCL6 and one or more Bcl2 family genes can be expressed from different vectors.

[0207] In one embodiment, there is a method of producing immune cells for adoptive cell therapy, comprising: (a) transducing or transfecting immune cells with (1) a heterologous CD30 protein, (2) a CD30 fusion protein, or (3) a combination of a heterologous BCL6 and one or more Bcl2 family genes, wherein the immune cells express (1) CD30, (2) a CD30 fusion protein, or (3) CD30, respectively; and (b) transducing or transfecting the immune cells with one or more therapeutic proteins. In certain embodiments, step (a) is performed before, simultaneously with, or after step (b). The transforming or transfecting in step (a) can be the same transforming or transfecting in step (b). In certain embodiments, (1), (2), or (3) are on the same vector as the therapeutic protein, but (1), (2), or (3) can be on a different vector from the therapeutic protein. In certain embodiments, following step (a), the immune cells from the method are analyzed for the presence of CD30 expressed on the surface of the immune cells, such as by flow cytometry, polymerase chain reaction, or a combination thereof. In some cases, the method further comprises administering the immune cells produced from the method to an individual in need thereof. In either case, the immune cells can be monitored in the individual, for example, using an agent that binds to CD30 (e.g., an antibody or an antibody-drug conjugate). In certain embodiments, the individual exhibits one or more adverse effects from the immune cells, and the individual is administered an effective amount of an agent that binds to CD30. The individual may exhibit toxicity from the immune cells, and the individual may be administered an effective amount of an agent that binds to CD30. The individual may exhibit graft-versus-host disease (GVHD) from the immune cells, and the individual may be administered an effective amount of an agent that binds to CD30. The individual may be one who no longer needs the immune cells, and the individual may be administered an effective amount of an agent that binds to CD30.

[0208] An embodiment of the present disclosure includes a method of reducing or preventing one or more adverse effects from a cell therapy in an individual, comprising targeting an extracellular domain of a chimeric polypeptide expressed on the surface of a cell of the cell therapy. The method may be further defined as administering to the individual an effective amount of one or more agents, such as an antibody (such as a monoclonal antibody) or an antibody-drug conjugate, that bind to the extracellular domain of the chimeric polypeptide expressed on the cell. Examples of adverse effects include GVHD, cytokine release syndrome, or immune effector cell-associated neurotoxicity syndrome. When the polypeptide comprises the extracellular domain of CD30, the expressed CD30 extracellular domain may or may not also comprise the entire CD30 protein. In certain cases, CD30 is a fragment of the entire CD30 protein that includes at least a portion of the extracellular domain. CD30 may be naturally expressed on immune cells. In some cases, CD30 is naturally heterologously expressed on immune cells. CD30 may be expressed on cells as a result of cells expressing heterologous BCL6 and one or more Bcl2 family genes. CD30 may be a CD30 fusion protein, such as one that includes at least a portion of the CD30 extracellular domain fused to an intracellular domain that includes endocytosis or internalization activity. Examples of intracellular domains include those derived from B-cell maturation antigen (BCMA), trop-2, CD317, CD3 gamma, CD4, CD79b, or combinations thereof. The CD30 fusion protein may include a CD30 transmembrane domain. In certain cases, the fusion protein includes SEQ ID NO: 47. The sequence encoding the fusion protein may include SEQ ID NO: 48.

[0209] In certain cases, prior to any targeting step, the individual's cells can be monitored in vivo, for example, using one or more agents that target the chimeric polypeptide. In certain embodiments, the agent that targets the chimeric polypeptide is an antibody that is used in a sufficiently low amount so as not to inhibit the therapeutic cells. The cell therapy can be allogeneic or autologous with respect to the individual. The cell therapy can include immune cells expressing one or more heterologous proteins, including therapeutic proteins, cytokines, fusions of cytokines and cytokine receptors, safety switches, or mixtures thereof. The therapeutic proteins can include one or more engineered antigen receptors, including chimeric antigen receptors, T cell receptors, or both, expressed by the cells. The cells can express chimeric polypeptides and chimeric antigen receptors.

[0210] An embodiment of the present disclosure includes a method of inhibiting a cell's activity, comprising exposing a cell transduced or transfected with a combination of heterologous BCL6 and one or more Bcl2 family genes to an effective amount of an agent that binds to CD30. In certain embodiments, inhibiting the activity is further defined as inducing apoptosis of the cell. The method may further comprise detecting binding of the agent to CD30 expressed on the cell. In certain cases, the method further comprises transducing or transfecting any type of cell encompassed herein with heterologous BCL6 and one or more Bcl2 family genes. The one or more Bcl2 family genes may be BCL2L1.

[0211] An embodiment of the present disclosure includes a method of inhibiting a cell's activity, comprising exposing a cell transduced or transfected with a CD30 fusion protein comprising at least a portion of the CD30 extracellular domain to an effective amount of an agent that binds to CD30. In certain embodiments, inhibiting the activity is further defined as inducing apoptosis of the cell. The method may further comprise detecting binding of the agent to CD30 expressed on the cell.

[0212] X. CD30 Positive Cells and Related Compositions CD30 is also known as TNFRSF8, D1S166E, Ki-1, tumor necrosis factor receptor superfamily member 8 and TNF receptor superfamily member 8. The present disclosure relates to compositions comprising CD30 positive cells that reduce the risk that cell therapy is toxic to a recipient individual and / or allow for monitoring of the production and / or location of the cells. CD30 positive cells produced using the methods of the present disclosure have a lower risk of toxicity compared to cells not produced using the methods of the present disclosure. Expression of the extracellular domain of the CD30 protein on the surface of a cell allows for targeting of the protein with one or more agents that recognize the domain on the surface and directly or indirectly result in inhibition of the cell, including cell death.

[0213] In certain embodiments, CD30 positive cells are utilized as a marker and safety switch for any type of cell, including cells that have the potential to become toxic to the recipient individual. Embodiments of the present disclosure include methods and compositions in which CD30 is effective as either or both a transduction marker and a safety switch. Cell therapy, in certain embodiments, includes CD30 positive cells, which are themselves immune cells that may or may not include modifications to express one or more heterologous genes.

[0214] CD30 positive cells include those in which CD30 is naturally expressed on the surface of the cell. Alternatively, cells may be modified by the hand of man to express some or all of CD30, and in some cases cells without such modifications will not express CD30. CD30 may or may not be wild type. In certain embodiments, some but not all of the CD30 protein is expressed within the cell, and when less than all of the CD30 protein is utilized, it includes at least a portion of the CD30 extracellular domain, if not all of the extracellular domain. In certain embodiments, the CD30 transmembrane domain is utilized, but not in other cases. In certain cases, at least a portion of CD30 is utilized in a fusion protein, including those in which the CD30 fusion protein is expressed on the surface of the cell. In such cases, the CD30 fusion protein includes at least a portion, if not all, of the CD30 extracellular domain. In certain cases, sufficient CD30 extracellular domain is included in the fusion protein so that an antibody that recognizes the CD30 extracellular domain can bind to it at the appropriate epitope.

[0215] Cells may also have the inherent ability to express CD30 after human modification, such as after transfection or transduction of one or more heterologous genes that are not CD30. For certain cells, including at least T cells (including □□ or □□), NK cells, NKT cells, monocytes, macrophages, B cells, mesenchymal stem cells (MSC) cells, hematopoietic stem cells (HSC), hematopoietic cells, induced pluripotent stem cells (iPSC) or derivatives thereof, mixtures thereof, derivatives thereof, where CD30 is not normally expressed, transfection or transduction of one or more heterologous genes results in expression of CD30. The heterologous genes may or may not be unrelated to the pathway utilizing CD30, and in certain cases, the heterologous genes are BCL6 and BCL2L1 genes or related genes. In such cases, this allows CD30 to be used as a transduction marker for successful transfection or transduction of cells with one or more heterologous genes.

[0216] CD30 (or derivatives thereof) as a membrane-bound protein may be utilized in a variety of therapeutic immune cell products. In some cases, the immune cells include at least T cells (including □□ or □□), NK cells, NKT cells, monocytes, macrophages, B cells, mesenchymal stem cells (MSC) cells, hematopoietic stem cells (HSC), hematopoietic cells, induced pluripotent stem cells (iPSC) or derivatives thereof, mixtures thereof, derivatives thereof, etc.

[0217] In certain cases, the membrane-bound protein comprises part or all of the extracellular domain of CD30 and / or the intracellular domain of CD30. In some cases, the membrane-bound protein is a full-length wild-type CD30 protein. CD30 in a cell may comprise the CD30 extracellular domain and the transmembrane domain, but not the CD30 intracellular domain. CD30 in a cell may comprise the CD30 extracellular domain and the CD30 intracellular domain, but not the CD30 transmembrane domain. Membrane-bound CD30 protein may comprise a cytoplasmic tail (having at least an internalization motif, meaning that the tail can interact with the clathrin-mediated intracellular trafficking complex to internalize anti-CD30 antibodies bound to CD30) that comprises one or more internalization cytoplasmic tails of one or more other membrane-bound receptors. Consensus sequences associated with clathrin-dependent endocytic sorting signals are known in the art (Traub, Molecular Cell Biology, Vol. 10, pp. 583-596, 2009). In some cases, the cytoplasmic tail is the cytoplasmic portion of a surface binding protein. In certain embodiments, the cytoplasmic tail is the cytoplasmic tail of CD30, BCMA, trop-2, CD317, CD3 gamma, CD4, CD79b, CD19, CD22, CD25, CD33, etc.

[0218] In one embodiment, CD30 is co-expressed in cells with one or more heterologous proteins in the cells. The heterologous protein may or may not be a therapeutic protein, including those that have therapeutic efficacy in themselves and / or confer therapeutic efficacy to the CD30-positive cells that express it. The cells can be manufactured to express CD30 and heterologous proteins at the same time or at different times. In some embodiments, cells that may or may not be CD30-positive are modified to express one or more heterologous proteins and then stored in a repository, including cryopreservation. If necessary, the cells can be thawed and further modified to express CD30, further modified to express CD30 fusion proteins, or transformed / transfected to express one or more heterologous genes, and then the expression of CD30 in the cells can be upregulated. In other cases, cells that express CD30, express a CD30 fusion protein, or are transformed / transfected to express one or more heterologous genes and then upregulate expression of CD30 in the cells are modified to express one or more heterologous proteins after thawing the cells from frozen storage in a repository.

[0219] In certain embodiments, for any CD30 positive cell, the heterologous gene may be one or more engineered antigen receptors, including chimeric antigen receptors or TCR expressing cells. In such cases, any engineered antigen receptor may target one or more antigens, including one or more cancer antigens. The engineered antigen receptor may target a single or multiple antigens of interest, including cancer antigens.

[0220] In certain cases, the CD30 positive cells are autologous or allogeneic (with respect to the individual) cells that may or may not express one or more xenogeneic proteins.

[0221] In some embodiments, CD30 positive cells express additional safety switches other than CD30 or CD30 fusion proteins, including herpes simplex virus thymidine kinase (HSV-tk) / ganciclovir, iCaspase9, tEGFR, synNotch, combinatorial target antigen recognition, inhibitory chimeric antigen receptors, and the like.

[0222] In some embodiments, a CD30 fusion protein is utilized that is a fusion of the CD30 extracellular domain with another protein fragment that includes a fragment that is at least a portion of the cytoplasmic region of a membrane-bound protein. As an example, the CD30 extracellular domain and transmembrane domain (PVLFWVILVLVVVGSSAFLL; SEQ ID NO:51) were fused to the B-cell maturation antigen (BCMA) cytoplasmic tail (underlined). The amino acid sequence of a representative CD30-BCMA fusion protein is as follows:

[0223] MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSV CPAGMIVKFPGTAQKNTVCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVS CSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSCARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR (SEQ ID NO: 47)

[0224] The DNA sequence of a representative CD30-BCMA fusion protein is as follows:

[0225]

[0226] In some embodiments, wild-type CD30 is utilized because it is naturally present on the cell, because it is a heterologous CD30 that has been transduced into the cell, or because it is expressed on the cell as a result of the transduction or transfection of one or more other genes into the cell.

[0227] Amino acid sequence:

[0228] (SEQ ID NO:49)

[0229] DNA sequence:

[0230]

[0231] Other specific fusion protein combinations are contemplated, including (1) CD30 extracellular antigen with trop-2 intracellular domain, which may or may not include a CD30 transmembrane domain, (2) CD30 extracellular antigen with CD317 intracellular domain, which may or may not include a CD30 transmembrane domain, (3) CD30 extracellular antigen with CD3 gamma intracellular domain, which may or may not include a CD30 transmembrane domain, (3) CD30 extracellular antigen with CD4 intracellular domain, which may or may not include a CD30 transmembrane domain, or (4) CD30 extracellular antigen with CD79b intracellular domain, which may or may not include a CD30 transmembrane domain. In embodiments where a CD30 transmembrane is not used, one of the following transmembrane domains may be utilized. The alpha, beta or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, or a DAP molecule, such as DAP10 or DAP12.

[0232] XI. Methods Related to CD30 Positive Cells The present disclosure relates to CD30 positive cells and uses thereof, including either native or recombinant CD30, including membrane-bound CD30 proteins that may or may not be chimeric with one or more other protein fragments. In certain embodiments, CD30 is utilized as: (1) a transduction and selection marker to enrich for therapeutic cell products during manufacturing; (2) a transduction marker to monitor infused cell products to assess in vivo proliferation, phenotype, function, trafficking and persistence; and (3) a safety switch to optionally eliminate infused therapeutic cells using monoclonal antibodies, antibody-drug conjugates or other approaches.

[0233] In certain cases, the disclosure encompasses methods in which CD30 is used as a transduction marker. For example, cells that may or may not express CD30 are transfected or transduced to express a protein having at least the CD30 extracellular domain, and cells that are candidates for successful transfection or transduction are assayed for the presence of the CD30 extracellular domain, for example, using one or more agents that bind to at least a portion of the extracellular domain. In some cases, the one or more agents are labeled so that this interaction can be detected, including detection by light, fluorescence, color, radioactivity, etc. In other cases, cells that may or may not express CD30 are transfected or transduced to express one or more proteins that are not CD30, but after each transfection or transduction, the cells express CD30 as a direct or indirect result of the transfection or transduction.

[0234] In some embodiments, CD30 protein is used as a selection marker. That is, a population of cells having at least one subset suspected to be CD30 positive is subjected to a method that allows the selection of CD30 positive cells, such as using a substrate that includes a CD30 binding agent on the surface. The population can be exposed under conditions sufficient to allow those CD30 positive cells in the population to bind to an agent, thereby eliminating cells that are not CD30 positive. The cells can then be released from the binding agent, such as after appropriate washing. In certain embodiments, the CD30 used as a selection marker is not endogenous CD30, but is expressed by cells for human hands, for example, after transfection or transduction with a heterologous CD30, a CD30 fusion protein, or one or more heterologous genes that are not CD30 but that result in its upregulation.

[0235] The disclosed methods also utilize CD30 or CD30-related derivative proteins (e.g., fusion proteins) to monitor cell products. Monitoring can be in vitro or in vivo, and monitoring can include assaying for cell proliferation, specific functions associated with the cells, and the like. In some cases, monitoring is in vivo, following administration of CD30-positive cells to a recipient individual, including administration by infusion. In certain cases, CD30-positive infused cell products allow for in vivo evaluation of various aspects of the infused cell therapy, including proliferation, phenotype, function, trafficking, and persistence. In some embodiments, monitoring CD30-positive cells provides information on the risk of the cell therapy becoming toxic to an individual. For example, CART cells may exhibit excessive proliferation after infusion, which can cause toxicity.

[0236] In certain embodiments, CD30 or CD30-related derivative proteins (e.g., fusion proteins) on cells are utilized as a safety switch to inhibit the activity of CD30-positive cells of cell therapy when the cells are no longer needed or when the individual shows one or more symptoms that the cell therapy is toxic to the individual or has become toxic to the individual over any particular period of time. The present methods and compositions are believed to be useful in reducing or preventing one or more adverse events such as cytokine release syndrome, neurotoxicity, immune effector cell-associated neurotoxicity syndrome, anaphylaxis / allergy, host rejection including at least GVHD, on-target on-tumor toxicity, and / or on-target / off-tumor toxicity (depletion of normal cells) in individuals who are at risk of having one or more symptoms, including imminent. The use of suicide genes or safety switches may be part of a planned protocol for treatment or may be used only when there is a recognized need for its use. In some cases, the safety switch delays the onset of toxicity and / or reduces the severity of toxicity, while in other cases, it prevents toxicity or inhibits toxicity altogether.

[0237] After infusion into an individual in need of therapeutic cells, the membrane-bound CD30 protein can be used to monitor the fate of the adoptively transferred T cells by flow cytometry, PCR, or other laboratory methods such as next generation sequencing, or using clinical trials such as imaging studies. In situations where the treatment results in adverse events such as cytokine release syndrome, GVHD, tumor formation, antibodies or antibody-drug conjugates or other approaches can be utilized to eliminate the infused cells in vivo.

[0238] The present disclosure provides membrane-bound CD30 proteins or fusion proteins that can be used, alone or in combination, as transduction and selection markers and / or as efflux safety switches. In some cases, cells express CD30 or CD30 fusion proteins that are used as safety switches and not as transduction or selection markers, while in other cases they are used as transduction or selection markers and not as safety switches.

[0239] The disclosed method also includes a method of inhibiting a cell's activity by targeting a surface protein on the cell, which comprises a part or all of the extracellular domain of CD30. The activity to be inhibited may be of any type, but in certain embodiments, the inhibition of activity is defined as inducing apoptosis of the cell. Exposing a cell to an effective amount of one or more agents that bind to CD30 may inhibit or slow the cell's growth, or may kill the cell. An agent that binds to CD30 may control the survival of the cell in any of the methods disclosed herein.

[0240] XII. Combination Therapy In certain embodiments, the compositions and methods of the present invention include an additional cancer treatment in the composition comprising the therapeutic cells. The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination thereof. The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0241] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0242] Immune cell therapy (in addition to the cell therapy of the present disclosure) may be administered in various combinations before, during, after, or compared to additional cancer therapy, such as immune checkpoint therapy. Administration may be at intervals ranging from the same time to minutes to days to weeks. In embodiments in which immune cell therapy is provided to a patient separately from the composition of the present disclosure, generally ensure that no significant period has elapsed between the time of each delivery so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the immunotherapy treatment and the disclosed composition may be provided to the patient within about 12-24 or 72 hours of each other, more specifically within about 6-12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period if several days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between the respective administrations.

[0243] Administration of any compound or cell therapy of the present embodiments to a patient will follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agents. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0244] A. Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to connote a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0245] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carboquone, metholedopa, and uredopa; ethylenimines and methylameramines, e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, K W-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongiostatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosfamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma I and calicheamicin omega I1); dynemicins (including dynemicin A); bisphosphonates such as clodronate; esperamicin;Also included are neocarzinostatin chromophore and related enediyne antibiotic chromophores, aclacinomycin, actinomycin, ausularmycin, azaserine, bleomycin, actinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidine-doxorubicin, and deoxydoxorubicin). sirolimusin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zavicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; fludarabine , 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergics such as mitotane and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enylic acid; Luracil; Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diazicon; Erformitin; Elliptinium acetate; Epothilone; Toglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitrelin; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin;Schizophyllan;spirogermanium;tenuazonic acid;triazicon;2,2',2"-trichlorotriethylamine;trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidin);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacitosine;arabinosides ("Ara-C");cyclophosphamide;taxoids such as paclitaxel and docetaxel gemcitabine;6-thioguanine;mercaptopurine;platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine;platinum;etoposide (VP -16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylchlornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0246] B. Radiation therapy Other agents that cause DNA damage and are widely used include what is commonly known as gamma radiation, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation (U.S. Patents. 5,760,395 and 4,870,287), and UV irradiation are also contemplated. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome construction and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0247] C. Immunotherapy Those skilled in the art will understand that additional immunotherapy (other than the disclosed cell therapy) may be used in conjunction or in combination with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may function alone as an effector of therapy or may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic drug, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells other than those with knockdown or knockout of TGF-beta R2.

[0248] Antibody-drug conjugates have emerged as a revolutionary approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for their antigen target with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells that bear concentrated levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The approval of two ADC drugs by the FDA, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has validated this approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets amenable to this approach and the generation of targeted MAbs. Two criteria for ADC targets are up-regulation / high levels of expression in tumor cells and strong internalization.

[0249] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer and immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0250] Examples of immunotherapies currently under investigation or in use include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as any type of interferon, IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0251] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either elevate signals (e.g., costimulatory molecules) or process signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0252] D.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0253] Removal of part or all of the cancerous cells, tissues or tumors may result in the formation of a cavity in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatments may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be in various dosages.

[0254] E. Other Agents It is contemplated that other agents can be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment.

[0255] XIII. Vector In certain embodiments, the cells encompassed herein have one or more vectors that can express any of the polypeptides encompassed herein.Various proteins can be delivered to recipient cells by any suitable vector, including viral vectors or non-viral vectors.Examples of viral vectors include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vectors.Examples of non-viral vectors include at least plasmid, transposon, lipid, nanoparticle, liposome, combinations thereof, etc.

[0256] When a cell is transduced with a vector encoding a chimeric polypeptide, if one or more other genes, such as, for example, a heterologous protein, are also required to be transduced into the cell, they may or may not be included in the same vector. Optionally, the chimeric polypeptide, the heterologous protein, etc. are expressed from the same vector molecule, such as the same viral vector molecule. In such a case, the expression of the polypeptides may or may not be regulated by the same regulatory elements. When the polypeptides are on the same vector, they may or may not be expressed as separate polypeptides. When they are expressed as separate polypeptides, they may be separated on the vector, for example, by a 2A element or an IRES element (or both types may be used once or multiple times on the same vector).

[0257] One of skill in the art would be well prepared to construct vectors by standard recombinant techniques (e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expression of the antigen receptors of the present disclosure.

[0258] A. Regulatory Elements The expression cassette contained in the vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked to a protein coding sequence, a splice signal with intervening sequences, and a transcription termination / polyadenylation sequence (5' to 3' direction). Promoters and enhancers that control the transcription of protein coding genes in eukaryotic cells can be composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information conveyed by each element, allowing different genes to evolve different, often complex patterns of transcriptional regulation. Promoters used in connection with the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the production of cancer therapy, the promoter can be effective under conditions of hypoxia.

[0259] B. Promoters / Enhancers The expression constructs provided herein include a promoter for driving the expression of any polypeptide. A promoter generally includes a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, separate elements that cover the start site itself help to anchor the location of initiation. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region upstream of the start site, but many promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame of the selected promoter (i.e., 3') is placed "downstream." The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0260] Spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, for example, spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0261] A promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are "non-naturally occurring", i.e., contain different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with the compositions disclosed herein. It is further contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0262] Of course, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cellular organelle, cell type, tissue, organ or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer and cell type combinations for protein expression (see, for example, Sambrook et al., 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0263] Additionally, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB, available over the World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0264] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as beta actin promoter, GADPH promoter, metallothionein promoter; and linked response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoter near minimal TATA box (tre). It is also possible to use a human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in GENBANK®, accession number X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from ATCC®, catalog number ATCC45007). In certain embodiments, the promoter is a CMVIE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, AdMLP, beta-actin, MHC class I or MHC class II promoter, although any other promoter useful for driving expression of therapeutic genes is applicable to the practice of the present disclosure.

[0265] In certain embodiments, the method of the present disclosure also relates to enhancer sequences, i.e. nucleic acid sequences that have the potential to increase the activity of a promoter and act in cis, regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter).However, enhancer function is not necessarily limited to such long distances, since it can function in the vicinity of a given promoter.

[0266] C. Initiation Signals and Linked Expression Specific initiation signals may also be used in the expression constructs provided in this disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG initiation codon. Those skilled in the art will be readily able to determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translation control signals and initiation codons may be either natural or synthetic. The efficiency of expression may be increased by including appropriate transcription enhancer elements.

[0267] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) as well as IRES from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic message. Thanks to the IRES element, each open reading frame is accessible to the ribosome for efficient translation. Multiple genes can be efficiently expressed by using a single promoter / enhancer to transcribe a single message.

[0268] As detailed elsewhere herein, certain 2A sequence elements can be used to create linked or co-expression of genes in constructs provided in the present disclosure. For example, cleavage sequences can be used to link open reading frames to form a single cistron to allow co-expression of genes. Exemplary cleavage sequences are Equine Rhinitis A Virus (E2A) or F2A (Foot and Mouth Disease Virus 2A) or "2A-like" sequences (e.g., Thosea asigna Virus 2A; T2A) or Porcine Teschovirus-1 (P2A). In certain embodiments, in a single vector, the multiple 2A sequences are not identical, but in alternative embodiments, the same vector utilizes two or more of the same 2A sequences. Examples of 2A sequences are provided in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety.

[0269] D. Origin of replication To propagate the vector in a host cell, it may contain one or more origin of replication sites (often called "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV described above, or an engineered oriP with a similar or enhanced function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, the origin of replication or autonomously replicating sequence (ARS) of other extrachromosomally replicating viruses described above can be used.

[0270] E. Selectable and Screenable Markers In some embodiments, the CD30 positive cells of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker would confer an identifiable change to the cell that allows easy identification of cells that contain the expression vector. In general, a selection marker is one that confers a property that allows selection. A positive selection marker is one whose presence allows its selection, and a negative selection marker is one whose presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0271] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, including screenable markers such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT), may be utilized. Those skilled in the art will likely also know how to use immunological markers in combination with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art.

[0272] XIV. Treatment Methods In various embodiments, the expression constructs, nucleic acid sequences, vectors, host cells, etc. contemplated herein, and / or pharmaceutical compositions comprising them, are used to prevent, treat, or ameliorate cancerous diseases, such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful, for example, for preventing, ameliorating, and / or treating cancer. An individual may utilize the treatment methods of the present disclosure as an initial treatment, or after (or in conjunction with) another treatment, such as after HSCT. Immunotherapy methods may be tailored to the needs of an individual with cancer based on the type and / or stage of the cancer, and in at least some cases, immunotherapy may be altered during the course of an individual's treatment.

[0273] In some embodiments, the present disclosure provides a method of immunotherapy comprising administering an effective amount of cells produced by the method of the present disclosure. In one embodiment, a medical disease or disorder is treated by the transfer of a cell population produced by the method of the present disclosure and which induces an immune response. In certain embodiments of the present disclosure, cancer is treated by the transfer of a cell population produced by the method of the present disclosure. Provided herein is a method of treating cancer or delaying the progression of cancer in an individual comprising administering to the individual an effective amount of a cell therapy. The method may be applied to the treatment of solid or hematological cancers.

[0274] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in solid or hematological tumors. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, and pancreatic cancer.

[0275] In certain embodiments of the present disclosure, the immune cells encompassed herein are delivered to an individual in need thereof, for example, an individual with cancer.In some cases, the individual is provided with one or more doses of immune cells.When the individual is provided with two or more doses of immune cells, the period between doses should be sufficient to allow time for proliferation in the individual, and in certain embodiments, the period between doses is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more.

[0276] In certain embodiments, the cells are administered in a therapeutically effective amount (between 1 cell and 10 cells) that ameliorates at least one symptom associated with cancer in an individual. 10 A therapeutically effective dose is provided to an individual in the range of 1 to 10 10 , 10~10 10 , 10 2 -10 10 , 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~108 , 10 3 ~10 7 , 10 3 ~10 6 , 10 3 ~10 5 , 10 3 ~10 4 , 10 4 ~10 10 , 10 4 ~10 9 , 10 4 ~10 8 , 10 4 ~10 7 , 10 4 ~10 6 , 10 4 ~10 5 , 10 5 ~10 10 , 10 5 ~10 9 , 10 5 ~10 8 , 10 5 ~10 7 , 10 5 ~10 6 , 10 6 ~10 10 , 10 6 ~10 9 , 10 6 ~10 8 , 10 6 ~10 7 , 10 7 ~10 10 , 10 7 ~10 9 , 10 7 ~10 8 , 10 8 ~10 10 , 10 8 ~10 9 Or 10 9 ~10 10In certain embodiments, an individual with cancer is provided one or more times with a therapeutically effective amount of specific therapeutic cells. In some embodiments, if the individual is at risk of adverse effects from these cells as a treatment, the concentration can be adjusted and / or there can be administration of one or more chimeric polypeptide agents that target antigens on the cells. In certain embodiments, an individual with cancer is provided one or more times with a therapeutically effective amount of specific immune cells, and then if the individual is at risk of adverse effects from the cells as a treatment, the concentration can be adjusted and / or there can be administration of one or more chimeric polypeptide agents that target antigens on the cells.

[0277] In certain embodiments, the vector can be stably integrated into the genome of the subject after administration. In certain embodiments, for example, viral vectors can be used that are specific to certain cells or tissues and persist intracellularly. Suitable pharmaceutical carriers and excipients are well known in the art. The composition prepared according to the present disclosure can be used for the prevention or treatment or delay of the above-identified diseases.

[0278] Furthermore, the present disclosure relates to a method for the prevention, treatment or amelioration of a neoplastic disease, comprising administering to a subject in need thereof an effective amount of the CD30 positive cells encompassed herein, the nucleic acid sequences, the vectors contemplated herein and / or the vectors produced by the methods contemplated herein.

[0279] The possible indications for administration of the exemplary cell composition are cancerous diseases, including any kind of neoplastic disease.The exemplary indications for administration of the CD30 positive cell composition are cancerous diseases, including any malignant tumor expressing a specific antigen, including those directed against CAR.Administration of the composition of the present disclosure is useful for all stages and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer.

[0280] Therapeutically effective amounts of the produced cells can be administered by several routes, including parenteral administration, e.g., intravenous, intraperitoneal, intramuscular, intrasternal, intratumor, intrathecal, intraventricular, reservoir, intraarticular injection, or infusion.

[0281] A therapeutically effective amount of cells produced for use in adoptive cell therapy is that amount that achieves a desired effect in the treated subject, for example, this may be the amount of immune cells necessary to inhibit progression or cause regression of a cancer.

[0282] The cell population produced can be administered in a treatment regimen consistent with the disease, for example, a single or several doses over one to several days to ameliorate the disease state, or regular doses over an extended period to inhibit disease progression and prevent disease recurrence. The precise dose employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective amount of cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dose that can be used to treat a human subject is at least 1×10 3 , at least 1 × 10 4 , 3.8×10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 T cells / m 2 In certain embodiments, the dose used to treat a human subject is in the range of about 3.8×10 9 ~Approx. 3.8×10 10 T cells / m 2 In a further embodiment, the therapeutically effective amount of T cells ranges from about 5×10 per kg of body weight. 6 ~ 7.5 x 10 cells per kg of body weight 8 cells, e.g., about 2 × 10 per kg of body weight 7~ approx. 5 x 10 cells 8 cells or approximately 5 x 10 per kg of body weight 7 ~ approx. 2 x 10 cells 8 The amount of T cells can vary from one cell to another. The exact amount of T cells can be easily determined by one skilled in the art based on the age, weight, sex and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0283] The present disclosure further includes co-administration protocols with other compounds that act through immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds. Clinical regimens for co-administration of the compounds of the present invention may include co-administration at the same time, before or after administration of other components. Particular combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0284] Embodiments relate to kits comprising a cell as defined herein, a construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein and / or a host as defined herein. The kits of the present disclosure are also contemplated to comprise a pharmaceutical composition as described herein above, alone or in combination with additional agents to be administered to an individual in need of medical treatment or intervention.

[0285] XV. Kits of the Present Disclosure Any of the compositions described herein may be included in a kit. In a non-limiting example, the kit includes a CD30 molecule, a cell that contains it, a vector that encodes a particular CD30 protein or a CD30 fusion protein, a vector that encodes a heterologous protein and / or a reagent for making it, any of which may be included in a suitable container means of the kit of the present disclosure. The kit may include immune cells, vectors, expression construct polynucleotides (viral or not) for insertion into the vector, and the like. Primers for amplification of any polynucleotide may be included. Optionally, the kit includes cryopreserved cells that include CD30 positive cells. Any reagents for transfection or transduction of cells may be included.

[0286] The compositions of the kits may be packaged in either aqueous media or lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which one or more components may be placed, preferably appropriately aliquoted. Where there is more than one component in the kit, the kit will generally also include a second, third or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in the vial. The kits of the present disclosure will also typically include a means for containing the molecules, cells encapsulating same, and / or reagents for making in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0287] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly contemplated. The composition may also be formulated into an injectable composition. In that case, the container means may itself be a syringe, pipette, and / or other such device from which the formulation may be applied to an infected area of ​​the body, injected into an animal, and / or even applied to and / or mixed with other components of the kit.

[0288] However, the components of the kit may also be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0289] Regardless of the number and / or type of containers, the kits of the present disclosure may also include and / or be packaged with instruments to aid in the injection / administration and / or placement of the final composition into the animal's body. Such instruments may be syringes, pipettes, forceps, and / or any such medically approved delivery vehicle. In some embodiments, reagents or devices or containers for ex vivo use are included in the kit. EXAMPLES

[0290] XVI. Examples The following examples are included to demonstrate certain embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute certain modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results, without departing from the spirit and scope of the invention.

[0291] Example 1 - BCMA extracellular domain expression on 293T cells 293T cells were transfected with a plasmid containing one of three different membrane-bound receptors, the BCMA extracellular domain fused to the PD-L1 hinge and transmembrane domain, the BCMA extracellular domain fused to the CD8α hinge and transmembrane domain, or full-length wild-type BCMA. The polypeptides are listed in Table 2 below. Each receptor was linked to GFP enhanced by the P2A peptide. One day after transfection, cells were stained with an APC-conjugated anti-BCMA monoclonal antibody (Figure 1). [Table 2]

[0292] Example 2 - Expression of BCMA ectodomain with or without internalization motifs in therapeutic T cells Primary T cells transduced with BCL6 and BCL2L1 were transduced with lentiviral vectors co-expressing CD19CAR and one of two different BCMA fusion constructs, the BCMA extracellular domain with the PD-L1 hinge and transmembrane domain but without a functional intracellular domain (shown in Figure 2, left panel), or the BCMA extracellular domain fused to the PD-L1 hinge and transmembrane domain and the cytoplasmic domain from Trop-2 (tBCMA; shown in Figure 2, middle panel). The polypeptides are listed in Table 3 below.

[0293] Cells were stained with APC-conjugated anti-BCMA antibody and recombinant FITC-labeled CD19-Fc protein (Figure 2). Results show that chimeric BCMA protein expression is consistent with CD19CAR expression, indicating that the chimeric BCMA protein can be used as a transduction marker for therapeutic T cells. [Table 3]

[0294] Example 3 - BCMA fusion proteins transduced into therapeutic T cells can be used to enrich T cells using anti-BCMA beads Primary T cells transduced with BCL6 and BCL2L1 were transduced as described in Example 2, stained with APC-conjugated anti-BCMA antibody, and then enriched using anti-APC magnetic beads. After 2 weeks of expansion, cells were stained with APC-conjugated anti-BCMA antibody and recombinant FITC-labeled CD19-Fc protein to confirm the purity of the transduced cells (Figure 3). The results show that the BCMA and anti-CD19CAR double positive population was significantly enriched by magnetic bead isolation.

[0295] Example 4 - Fusion proteins containing the Trop2 cytoplasmic domain as a safety switch to eliminate T cells Primary T cells transduced with BCL6 and BCL2L1 were transduced with BCMA fusion proteins, concentrated as described in Example 3, and cultured in the absence or presence of belantamab mafodotin (an antibody-drug conjugate targeting BCMA) at concentrations of 0 μg / mL, 5 μg / mL, or 20 μg / mL. On day 2, viable cells were counted by flow cytometry using counting beads, and the percent change in viable cells with belantamab mafodotin compared to no drug was calculated (FIG. 4). The results show that therapeutic T cells expressing BCMA with the Trop-2 intracellular domain (tBCMA) were highly sensitive to the cytotoxic activity of belantamab mafodotin.

[0296] Example 5 - Expression of BCMA extracellular domain with or without the cytoplasmic tail on Jurkat cells Jurkat cells were transduced with lentiviral vectors expressing the BCMA ectodomain alone, the BCMA ectodomain fused to the cytoplasmic domain from CD317, or the BCMA ectodomain fused to the cytoplasmic domain from CD3γ. The polypeptides are listed in Table 4 below.

[0297] Cells were cultured in the absence or presence of belantamab mafodotin at concentrations of 25 μg / mL or 12.5 μg / mL. After 4 days, the medium was replaced with fresh medium without belantamab. After an additional 7 days of culture, the percentage of BCMA-positive cells was determined by flow cytometry (Figure 5). The results show a dose-dependent decrease in the percentage of BCMA-positive cells, indicating specific killing of BCMA-positive cells by belantamab. [Table 4]

[0298] Example 6 - CD30 as a safety switch CD30 expression on T cells can be induced or maintained on T cells by overexpression of BCL6 and BCL2L1 genes. BCL6 and BCL2L1 genes were transfected into primary T cells. Expression of CD30 and CD69 was measured by flow cytometry (Figure 6). CD30 was constitutively expressed in both αβ and γδ T cells (>99% of cells). In contrast, about 15% of primary T cells cultured in vitro for 4 weeks expressed CD30. Thus, CD30 can be used as a selection marker during production, as a transduction marker for monitoring in patients after infusion, and as a safety switch for elimination of therapeutic cells in case of severe adverse events.

[0299] Example 7 - Use of CD30 as a safety switch in therapeutic T cells expressing CD30 CD30-transduced primary T cells expressing BCL6 and BCL2L1 were cultured in the absence or presence of increasing concentrations of brentuximab vedotin. Raji Burkitt's lymphoma tumor cells, which do not express CD30, were used as a control. Cells were harvested, stained with live / dead stain, and absolute viable cell numbers were determined by flow cytometry using counting beads on days 1, 2, 3, and 4. The percent change in viable cell count was determined compared to cells cultured in the absence of brentuximab and is shown in the graph in Figure 7. The data suggest efficient killing (up to 95%) of CD30-expressing T cells compared to control Raji cells. Nonspecific cytotoxicity was observed at higher concentrations and longer incubations.

[0300] Example 8 - CD30 extracellular domain fused to the BCMA cytoplasmic tail can be expressed on 293T cells 293T cells were transduced with a lentiviral vector expressing both CD19CAR and Her2 domain 4 and CD30 extracellular domain fused to BCMA cytoplasmic tail (diagram shown in FIG. 8). Polypeptides are listed in Table 5 below. Transduced 293T were stained with both anti-CD30 and AF647-conjugated trastuzumab (FIG. 9). Transduced 293T cells were cultured in the presence of different concentrations of brentuximab for 4 days, and the cell number changes of both Her2-positive (also expected to be CD30-positive) and Her2-negative (also expected to be CD30-negative) 293T cells were counted using flow counting beads on day 4 (FIG. 9, right panel). The results showed that the viable cell number of CD30-positive (Her2-positive) 293T cells was significantly reduced compared to the control. [Table 5]

[0301] Example 9 - Jurkat cells expressing Her2 domain 4 can be killed by CAR-T cells. Jurkat T cells were transduced with lentiviral vectors expressing both CD19CAR and Her2 and truncated EGFR (diagram shown in FIG. 10). Polypeptides are listed in Table 6 below. Transduced cells were killed in the presence of trastuzumab and CD16-expressing CAR-T cells (FIG. 11A-FIG. 11B). FIG. 11A shows the expression of CD19CAR and Her2 domain 4 detected by staining with CD19-Fc fusion protein and AF647-conjugated trastuzumab, respectively. FIG. 11B shows the percent change in viable Jurkat cells when co-cultured with CD16-expressing CAR-T in the presence of trastuzumab. [Table 6]

[0302] Example 10 - Trop2 as a safety switch 293T cells were transfected with lentiviral plasmids containing anti-CD19CAR, Her2 domain IV and Trop2 genes (diagram shown in FIG. 12) using LIPOFECTAMINE™ 3000. Polypeptides are listed in Table 7 below. Flow cytometry analysis was performed 24 hours after transfection (FIG. 13). Data shows co-expression of anti-CD19CAR and Trop2 in transfected cells. [Table 7]

[0303] Example 11 - Domain 3 and partial domain 4 of EGFR as a safety switch 293T cells were transfected with a lentiviral plasmid containing both CD19CAR and Her2 and truncated EGFR (EGFR domain 3-partial domain 4 and CD8 hinge and TM; construct shown in FIG. 14). The sequence of truncated EGFR (EGFR domain 3-partial domain 4 and CD8 hinge and TM) is shown in Table 8 below. The data shows co-expression of anti-CD19CAR and truncated EGFR in transfected cells detected by CD19-Fc fusion protein and AF647-conjugated cetuximab (FIG. 15, right panel). The left panel of FIG. 15 shows untransduced 293T cells. [Table 8]

[0304] Example 12 - tBCMA safety switch has both in vitro and in vivo efficacy Primary T cells transduced with BCL6 and BCL2L1+ / -CD19CAR and expressing truncated BCMA (tBCMA) were cultured in the absence or presence of increasing concentrations of the anti-BCMA antibody drug conjugate belantamab mafodotin. Raji Burkitt lymphoma tumor cells, which do not express BCMA, were used as a control. Cells were harvested, stained with live / dead stain, and absolute viable cell numbers were determined by flow cytometry using counting beads on day 2. Percent change in viable cell count was determined compared to cells cultured in the absence of brentuximab and is shown in the graph. The data suggest efficient killing (up to 80%) of BCMA-expressing T cells compared to control Raji cells. Nonspecific cytotoxicity was observed at higher concentrations.

[0305] Primary T cells transduced with BCL6, BCL2L1, CD19CAR, tBCMA, luciferase and IL-15 were injected into NSG mice IV via the tail vein on day 0 (2 × 10 6 Belantamab mafodotin was injected IV via the tail vein at a dose of 2.5 mg / kg body weight into mice 3 and 4 on day 3, and into mice 1 and 2 on day 11. T cell proliferation and persistence was monitored by bioluminescence imaging at the indicated time points. The data show that T cells can be very efficiently eradicated by belantamab mafodotin when injected before (mice 3 and 4) or after (mice 1 and 2) robust proliferation in vivo.

[0306] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. (a) an extracellular domain derived from B-cell maturation antigen (BCMA); (b) a hinge region derived from programmed cell death 1 ligand 1 (PDL1); and (c) a transmembrane domain; and (d) an intracellular domain; and A chimeric polypeptide comprising:

2. The chimeric polypeptide of claim 1, wherein the extracellular domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

19.

3. The chimeric polypeptide of claim 2 , wherein the extracellular domain comprises SEQ ID NO:

19.

4. The chimeric polypeptide of claim 3, wherein the extracellular domain consists of SEQ ID NO:

19.

5. The chimeric polypeptide of claim 1 , wherein the hinge region comprises SEQ ID NO:

23.

6. The chimeric polypeptide of claim 5 , wherein the hinge region consists of SEQ ID NO:

23.

7. 2. The chimeric polypeptide of claim 1, wherein the transmembrane domain is a transmembrane domain derived from the alpha or beta chain of a T cell receptor, or from CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154.

8. The chimeric polypeptide of claim 1 , wherein the transmembrane domain is a transmembrane domain derived from CD8α.

9. 2. The chimeric polypeptide of claim 1, wherein the transmembrane domain is a transmembrane domain derived from PDL1.

10. The chimeric polypeptide of claim 9, wherein the transmembrane domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

25.

11. The chimeric polypeptide of claim 10 , wherein the transmembrane domain comprises SEQ ID NO:

25.

12. The chimeric polypeptide of claim 1, wherein the intracellular region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

40.

13. The chimeric polypeptide of claim 12, wherein the intracellular region comprises SEQ ID NO:

40.

14. The chimeric polypeptide of claim 13, wherein the intracellular region consists of SEQ ID NO:

40.

15. The chimeric polypeptide of claim 1 , wherein the chimeric polypeptide does not contain a signaling domain.

16. The chimeric polypeptide of claim 1 , wherein the chimeric polypeptide does not contain an intracellular region derived from BCMA.

17. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

1.

18. The chimeric polypeptide of claim 17, wherein the chimeric polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

1.

19. The chimeric polypeptide of claim 18 , wherein the chimeric polypeptide comprises SEQ ID NO:

1.

20. A chimeric polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

1.

21. A chimeric polypeptide comprising SEQ ID NO:

1.

22. A chimeric polypeptide consisting of sequence number 1.

23. The chimeric polypeptide of claim 1, wherein the chimeric polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

38.

24. The chimeric polypeptide of claim 23 , wherein the chimeric polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

38.

25. The chimeric polypeptide of claim 24, wherein the chimeric polypeptide comprises SEQ ID NO:

38.

26. A chimeric polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

38.

27. A chimeric polypeptide comprising SEQ ID NO:

38.

28. A chimeric polypeptide consisting of sequence number 38.

29. A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric polypeptide of claim 1.

30. A vector comprising the nucleic acid molecule of claim 29.

31. A method for producing an engineered cell, comprising introducing into a cell a chimeric polypeptide according to any one of claims 1 to 28, a nucleic acid molecule according to claim 29 or a vector according to claim 30.

32. An engineered cell comprising a chimeric polypeptide according to any one of claims 1 to 28, a nucleic acid molecule according to claim 29 or a vector according to claim 30.

33. 33. The engineered cell of claim 32, wherein the engineered cell is a T cell.

34. 34. The engineered cell of claim 33, wherein the T cell is a CD4+ T cell, a CD8+ T cell, an iNKT cell, an NKT cell, a γδT cell, or a regulatory T cell.

35. 33. The engineered cell of claim 32, wherein the engineered cell is a natural killer cell.

36. 33. The engineered cell of claim 32, wherein the engineered cell is an induced pluripotent stem cell (iPSC).

37. 33. The engineered cell of claim 32, wherein the engineered cell is an iPSC-derived cell.

38. 33. The engineered cell of claim 32, further comprising a chimeric antigen receptor (CAR).

39. 39. The engineered cell of Claim 38, wherein the chimeric polypeptide is operably linked to the CAR.

40. 33. The engineered cell of claim 32, further comprising a T cell receptor (TCR).

41. The engineered cell of claim 40, wherein the chimeric polypeptide is operably linked to the TCR.

42. 33. A population of engineered cells comprising the engineered cell of claim 32.

43. 33. A method for detecting, isolating, depleting or purifying the engineered cells of claim 32, comprising contacting the engineered cells with a BCMA-binding protein.

44. 44. The method of claim 43, wherein the BCMA binding protein is an anti-BCMA antibody or an antigen-binding fragment thereof.

45. 44. The method of claim 43, further comprising separating the cell from a population of cells using the BCMA binding protein.

46. 44. The method of claim 43, further comprising detecting the cell with an imaging agent, wherein the BCMA binding protein is linked to the imaging agent.

47. 44. The method of claim 43, wherein the BCMA binding protein is linked to a cytotoxic agent.

48. 48. The method of claim 47, wherein the BCMA binding protein linked to a cytotoxic agent is belantamab mafodotin.

49. 44. The method of claim 43, wherein contacting the engineered cell with the BCMA binding protein is performed in vitro.

50. 44. The method of claim 43, wherein contacting the engineered cell with the BCMA binding protein is performed ex vivo.

51. 44. The method of claim 43, wherein contacting the engineered cell with the BCMA binding protein occurs in vivo.

52. A chimeric polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

3.

53. 1. A method for detecting, isolating, depleting or purifying a cell, the method comprising: (a) an extracellular domain derived from B-cell maturation antigen (BCMA); (b) a hinge region derived from programmed cell death 1 ligand 1 (PDL1); and (c) a transmembrane domain; and (d) an intracellular domain; The method comprises contacting the cell with a BCMA binding protein.

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