BCMA-targeted CAR-T cell therapy for multiple myeloma

Abstract

1. A method for assessing a subject's responsiveness to a therapy comprising T cells expressing a bivalent BCMA-targeting chimeric antigen receptor (CAR), the method comprising administering the T cells to the subject and assessing the subject's responsiveness to the therapy based on the length of time the subject remains minimal residual disease (MRD) negative.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 421,740, filed November 2, 2022, the disclosure of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference. The Sequence Listing XML file submitted herewith is entitled "14651-049-228_SEQ_LISTING.xml", was created on October 16, 2023, and is 28,402 bytes in size.

[0003] Provided herein are methods for assessing the responsiveness of a subject (e.g., a subject with multiple myeloma) to a therapy comprising T cells expressing a bivalent BCMA-targeting chimeric antigen receptor (CAR). [Background technology]

[0004] Multiple myeloma (MM), classified as a B-cell neoplasm, is an incurable, aggressive plasma malignancy that grows uncontrollably in the bone marrow, disrupting the normal metabolic production of blood cells and causing painful bone lesions (Garfall, AL et al., Discovery Med. 2014, 17, 37). Clinically, multiple myeloma can present with hypercalcemia, renal failure, anemia, bone lesions, bacterial infection, hyperviscosity, and amyloidosis (Robert Z. Orlowski, Cancer Cell. 2013, 24(3)). Cellular immunotherapy based on antibodies targeting B-cell maturation antigen (BCMA) has recently demonstrated substantial clinical benefit for patients with multiple myeloma. However, the art needs meaningful clinical endpoints to effectively evaluate patient response to such cellular immunotherapy. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, provided herein is a method for assessing a subject's responsiveness to a therapy comprising T cells expressing a bivalent BCMA-targeting chimeric antigen receptor (CAR), the method comprising: (a) administering the T cells to the subject; (b) measuring the length of time the subject maintains minimal residual disease (MRD)-negative status; and (c) assessing the subject's responsiveness to the therapy based on: (i) maintenance of MRD-negative status for less than 6 months; (ii) maintenance of MRD-negative status for at least 6 months and less than 12 months; or (iii) maintenance of MRD-negative status for at least 12 months.

[0006] In some embodiments, the bivalent BCMA-targeting CARs provided herein comprise an extracellular antigen-binding domain comprising a first VHH domain and a second VHH domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the first VHH domain comprises CDR1, CDR2, and CDR3 as set forth in the VHH domain comprising the amino acid sequence of SEQ ID NO: 2, and the second VHH domain comprises CDR1, CDR2, and CDR3 as set forth in the VHH domain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 18, CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and the second VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first VHH domain comprises the amino acid sequence of SEQ ID NO:2 and the second VHH domain comprises the amino acid sequence of SEQ ID NO:4.

[0007] In some embodiments, the first VHH domain is N-terminal to the second VHH domain, or the first VHH domain is C-terminal to the second VHH domain.

[0008] In some embodiments, the first VHH domain is linked to the second VHH domain via a linker comprising the amino acid sequence of SEQ ID NO:3.

[0009] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0010] In some embodiments, the transmembrane domain is derived from CD8α and comprises the amino acid sequence of SEQ ID NO:6.

[0011] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ comprising the amino acid sequence of SEQ ID NO:8.

[0012] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and any combination thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD137 comprising the amino acid sequence of SEQ ID NO:7.

[0013] In some embodiments, the CARs provided herein further comprise a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α comprising the amino acid sequence of SEQ ID NO:5.

[0014] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide, hi some embodiments, the signal peptide is derived from CD8α comprising the amino acid sequence of SEQ ID NO:1.

[0015] In some embodiments, the CAR provided herein comprises the amino acid sequence of SEQ ID NO: 17.

[0016] In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is refractory or relapsed multiple myeloma.

[0017] In some embodiments, the methods provided herein include obtaining a bone marrow aspirate or biopsy from a subject to assess MRD status. In some embodiments, MRD status is monitored using next-generation sequencing (NGS) of bone marrow aspirate DNA. In some embodiments, NGS is performed via clonoSEQ.

[0018] In some embodiments, a baseline bone marrow aspirate is used to define the myeloma clone, and a post-treatment sample is used to assess MRD negativity. In some embodiments, an evaluable sample has passed one or more, or all, of calibration, quality control, and sufficiency of evaluable cells at a particular sensitivity level. In some embodiments, the sensitivity level is about 10 -6 , 10 -5 , 10 -4 , or 10 -3 is.

[0019] In some embodiments, the length of time that a subject remains MRD-negative is measured from the time that MRD is first achieved in the subject.

[0020] In some embodiments, the methods provided herein include assessing the likelihood that a subject will have a complete response (CR), a partial response (PR), a stringent CR (sCR), or very good PR (VGPR).

[0021] In some embodiments, the methods provided herein include determining that the subject is likely to have CR, PR, sCR, or VGPR if the MRD-negative status is maintained in the subject for less than six months. In some embodiments, the methods provided herein include determining that the subject is likely to have CR or sCR if the MRD-negative status is maintained in the subject for more than six months.

[0022] In some embodiments, the methods provided herein include assessing the likelihood of a subject having progression-free survival.

[0023] In some embodiments, the methods provided herein include determining that if the MRD-negative status is maintained in the subject for longer than 6 months, the subject is likely to have progression-free survival for at least 12 months after treatment.

[0024] In some embodiments, the methods provided herein include determining that if the MRD-negative status is maintained in the subject for greater than 12 months, the subject is likely to have progression-free survival for at least 24 months following treatment.

[0025] In some embodiments, the methods provided herein include assessing duration of response in the subject.

[0026] In some embodiments, the methods provided herein include determining that if MRD-negative status is maintained in a subject for longer than 6 months, the subject is likely to have a duration of response greater than 12 months.

[0027] In some embodiments, the methods provided herein include determining that if MRD-negative status is maintained in a subject for greater than 12 months, the subject is likely to have a duration of response greater than 24 months. [Brief explanation of the drawings]

[0028] [Figure 1]This figure shows the expression of BCMA antigen on the surface of GCs, memory and plasmablasts in lymph nodes, long-surviving plasma cells in bone marrow LN and MALT, and multiple myeloma cells. BAFF-R antigen is not expressed on plasmablasts, long-surviving plasma cells, or multiple myeloma cells. TACI is expressed on memory and plasmablasts, long-surviving plasma cells, and multiple myeloma cells. CD138 is expressed only on long-surviving plasma cells and multiple myeloma cells.

[0029] [Figure 2]

[0033] Figure 1 shows the design of a siltacabtagene autoleucel CAR. Siltacabtagene autoleucel contains two VHH domains, in contrast to the single VL and single VH domains found on various other CARs. Siltacabtagene autoleucel contains intracellular CD137 and human CD3 zeta domains.

[0030] [Figure 3] A schematic diagram is shown for the preparation of virus encoding the siltacac butagen autoleucel CAR, transduction of patient-derived T cells with the virus, and subsequent preparation of CAR T cells expressing siltacac butagen autoleucel.

[0031] [Figure 4] The demographics and disease characteristics of the patient population in the Phase 1b portion of the study are summarized.

[0032] [Figure 5]A schematic diagram of the study design for siltacabtagene Autoreucel CAR T cells is shown. The patient population includes those with relapsed or refractory multiple myeloma with three prior lines or dual resistance to a PI / IMiD and previous PI, IMiD, and anti-CD38 exposure. The primary objective is safety and establishment of RP2D, including testing the incidence and severity of adverse events (Phase 1b). Another primary objective is efficacy: ORR-PR or better as defined by the IMWG (Phase 2). The following are secondary objectives: incidence and severity of adverse events (Phase 2), and any further efficacy characterization.

[0033] [Figure 6] The development and duration of cilta-cel as measured by blood levels is shown.

[0034] [Figure 7] Progression-free survival outcomes for patient subgroups based on MRD-negative status are shown. MRD-negative < 6 months: Patients who achieved MRD-negative but became MRD-positive less than 6 months later. MRD-negative ≥ 6: Patients who achieved MRD-negative but became MRD-positive more than 6 months but less than 12 months later. MRD-negative ≥ 12 months: Patients who achieved MRD-negative and remained MRD-negative for 12 months or more. MRD-positive: Patients who did not achieve MRD-negative at any time point. The top plot shows censored observations in the corresponding patient subgroup. The x-axis shows the time in months that patients in each subgroup remain alive and progression-free. The y-axis represents the percentage of patients in each subgroup who are alive and progression-free after the given length of time indicated by the x-axis. The table below the plot shows the number of patients in each subgroup who remain progression-free and alive at each time point, aligned with the 3-month intervals indicated on the x-axis of the plot.

[0035] [Figure 8]Duration of response is shown for patient subgroups based on MRD-negative status. MRD-negative < 6 months: Patients who achieved MRD-negative but became MRD-positive less than 6 months later. MRD-negative ≥ 6 months and < 12 months: Patients who achieved MRD-negative but became MRD-positive more than 6 months but less than 12 months later. MRD-negative ≥ 12 months: Patients who achieved MRD-negative and remained MRD-negative for 12 months or more. MRD-positive: Patients who did not achieve MRD-negative at any time point. The top plot represents censored observations in the corresponding patient subgroup. The x-axis indicates the time in months that responding patients (responders) in each subgroup remain alive and progression-free (i.e., patients who continued to respond). The y-axis represents the percentage of responders in each subgroup who are alive and progression-free after the given length of time indicated by the x-axis. The table below the plots shows the number of responders in each subgroup who are still responding at each time point, aligned by the 3-month intervals indicated on the x-axis of the plot.

[0036] [Figure 9] Key patient and disease characteristics in these different MRD-negative groups are shown. a ≥1 PI, ≥1 IMiD, and 1 anti-CD38 antibody. b ≥2 PIs, ≥2 IMiDs, and 1 anti-CD38 antibody. ECOG, Eastern Cooperative Oncology Group; IMiD, immunomodulatory agent; PI, proteasome inhibitor.

[0037] [Figure 10] Responses in MRD-positive patients are shown. Patients who did not achieve MRD negativity at any time point were considered MRD-positive (n=5). The y-axis lists de-identified patient numbers. The x-axis shows time in months.

[0038] [Figure 11]Response to cilta-cel in MRD subgroups is shown. All patients with persistent MRD negativity for ≥6 months achieved sCR. aORR=sCR+CR+VGPR+PR; may not sum properly due to rounding. DOR, duration of response; MRD, minimal residual disease; ORR, overall response rate; PR, partial response; sCR, durable complete response; VGPR, best partial response.

[0039] [Figure 12] Progression-free survival outcomes are shown for patient subgroups based on MRD-negative status. MRD-negative < 6 months: patients who achieved MRD-negativity but became MRD-positive after less than 6 months. MRD-negative ≥ 6: patients who achieved MRD-negativity but became MRD-positive after more than 6 months but less than 12 months. MRD-negative ≥ 12 months: patients who achieved MRD-negativity and remained MRD-negative for 12 months or more. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure is based, in part, on the surprising discovery of a correlation between the length of time a subject maintains a minimal residual disease (MRD)-negative state and the subject's responsiveness (as measured by various techniques) to treatment with engineered T cells expressing a bivalent BCMA-targeted CAR. Accordingly, the length of time a subject maintains an MRD-negative state can be used to determine the subject's responsiveness to treatment with engineered T cells expressing a bivalent BCMA-targeted CAR (such as "siltacabtagene autoleucel" or "cilta-cel").

[0041] Some aspects of the present disclosure are described below with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present disclosure. However, one of ordinary skill in the art will readily recognize that the present disclosure can be practiced without one or more of the specific details or can be practiced using other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited by the illustrated order of acts or events, as some acts may be performed in a different order and / or simultaneously with other acts or events. Furthermore, not all illustrated acts, steps, or events are required to implement a methodology in accordance with the present disclosure.

[0042] Unless otherwise defined, all technical terms, notations, and other scientific or scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Furthermore, it will be understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0043] 5.1.Definition The techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed by those of skill in the art using conventional methodologies, such as the widely used methodologies described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Duebel eds., 2nd ed. 2010). Unless otherwise defined herein, scientific and technical terms used in this description have the meanings commonly understood by those of skill in the art. For the purposes of interpreting this specification, the following explanations of terms shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any explanation of a term provided contradicts any material incorporated herein by reference, the explanation of the term set forth below shall prevail.

[0044] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof (e.g., domain antibodies) as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species, such as mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells that are capable of binding to a specific molecular antigen and are within the immunoglobulin class of polypeptides composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 amino acids or more and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those derived from Camelidae species (e.g., llamas or alpacas), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above (which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived). Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.Specifically, the antibodies provided herein include immunoglobulin molecules and molecules containing an immunologically active portion of an immunoglobulin molecule, such as an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). For such antibody fragments, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plueckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody. The antibody can be neither an agonist antibody nor an antagonist antibody.

[0045] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0046] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2, and CH3. The constant region includes a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0047] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in four-chain antibodies. Camelids (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0048] "Single domain antibody" or "sdAb," as used herein, refers to a single monomeric variable antibody domain and has antigen-binding capability (e.g., a single domain antibody that binds to BCMA). Single domain antibodies comprise a VHH domain as described herein. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains, such as those derived from Camelidae species (e.g., llamas), single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. For example, single domain antibodies may be derived from antibodies raised in Camelidae species as described herein, such as camel, llama, dromedary, alpaca, and guanaco. Other species outside of Camelidae may produce heavy chain antibodies that naturally lack light chains; VHHs derived from such other species are within the scope of this disclosure. In some embodiments, a single domain antibody (e.g., VHH) provided herein has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody can be genetically fused or chemically conjugated to another molecule (e.g., a drug) as described herein. A single domain antibody can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0049] The terms "bind" or "binding" refer to interactions between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the overall non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The association rate (k on ) to the dissociation rate (k off ) ratio (k off / k on ) is the dissociation constant K D which is inversely proportional to the affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies depending on the antibody-antigen complex. on and k off The dissociation constant K of the antibodies provided herein depends on both D can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the likelihood of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0050] In the context of the binding molecules described herein, terms such as "binds to," "specifically binding to," and similar terms are also used interchangeably herein to refer to binding molecules, such as polypeptides, of an antigen-binding domain that specifically binds to an antigen. Binding molecules or antigen-binding domains that bind or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those skilled in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise and may be more than 10 times the background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent to which a binding molecule or antigen-binding domain binds to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen-binding domains that bind to antigens include those that have the ability to bind to antigens with sufficient affinity to make them useful for targeting antigens, e.g., as therapeutic and / or diagnostic agents. In certain embodiments, binding molecules or antigen-binding domains that bind to an antigen have a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. D In certain embodiments, the binding molecule or antigen-binding domain binds to an epitope that is conserved among antigens of different species.

[0051] In certain embodiments, a binding molecule or antigen-binding domain can comprise a "chimeric" sequence, in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences can include humanized sequences.

[0052] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a non-human (e.g., camelid, murine, non-human primate) antibody in a "humanized" form comprising sequences from a human immunoglobulin (e.g., recipient antibody), in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. Such modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0053] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "fully human antibody" or "human antibody," where these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. A binding molecule can comprise a single-domain antibody sequence. In specific embodiments, these terms refer to an antibody comprising variable and constant regions of human origin. A "fully human" antibody can also encompass, in certain embodiments, an antibody that binds a polypeptide and that is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human and / or an antibody produced using human antibody production techniques. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)).For the preparation of human monoclonal antibodies, the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) can also be used. Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been modified to produce such antibodies in response to antigen challenge but render their endogenous gene loci incompetent (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Brueggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584, regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006), regarding human antibodies generated by human B-cell hybridoma technology.

[0054] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cows) transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and closely related to human germline VH and VL sequences, are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0055] In certain embodiments, a binding molecule or antigen-binding domain can comprise a portion of a "monoclonal antibody," which term, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in small amounts, or well-known post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation, such that each monoclonal antibody typically recognizes a single epitope on the antigen. In a specific embodiment, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method of making the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or can be made using recombinant DNA methods in bacteria or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies they express are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0056] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al., supra; Nick Deschacht et al., J Immunol 2010;184:5696-5704). Chothia instead refers to the position of structural loops (see, for example, Chothia and Lesk, J. Mol. Biol.196:901-17(1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Duebel eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Here, CDRs are referenced both in terms of amino acid sequence and location within the light or heavy chain.Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides in structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin onto an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plueckthun, J. Mol. Biol. 309:657-70 (2001). Correspondence between numbering systems, including, for example, Kabat numbering and the IMGT proprietary numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are illustrated in the table below. [Table 1]

[0057] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof, such as a variable region, and individual CDRs of the antibody or region thereof (e.g., CDR-H1, CDR-H2) should be understood to encompass the complementarity-determining regions as defined by any of the known schemes described hereinabove. In some instances, schemes for identifying a particular CDR or CDRs are designated, such as CDRs as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR is provided. It should be noted that CDR regions can also be defined by a combination of various numbering schemes, such as a combination of the Kabat and Chothia numbering schemes or a combination of the Kabat and IMGT numbering schemes. Thus, terms such as "a CDR as shown in a particular VH or VHH" include, but are not limited to, any CDR1 as defined by the exemplary CDR numbering schemes described above. One of skill in the art will appreciate that given a variable region (eg, VHH, VH, or VL), the CDRs within that region may be defined by different numbering systems or combinations thereof.

[0058] The hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

[0059] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains, which are not directly involved in binding to an antigen by an antibody but exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portions of the immunoglobulin, the variable region, which contain the antigen-binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0060] The term "framework" or "FR" refers to those variable domain residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0061] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of the polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more noncontiguous regions of the polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). Those skilled in the art will generally understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether those amino acids are folded into a native three-dimensional protein structure. In other embodiments, the amino acid residues that make up the epitope must adopt a particular conformation (eg, bend, twist, turn, or fold) in order for the binding molecule to recognize and bind to the epitope.

[0062] "Percent (%) amino acid sequence identity" and "homology," in the context of peptide, polypeptide, or antibody sequences, are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences under comparison.

[0063] The term "specificity" refers to the selective recognition of an antigen-binding protein (such as a CAR or sdAb) for a specific epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecificity," as used herein, means that an antigen-binding protein (such as a CAR or sdAb) has two or more antigen-binding sites, at least two of which bind to different antigens. "Bispecificity," as used herein, means that an antigen-binding protein (such as a CAR or sdAb) has two different antigen-binding specificities. The term "monospecific" CAR, as used herein, means an antigen-binding protein (such as a CAR or sdAb) with one or more binding sites that each bind to the same antigen.

[0064] The term "valent," as used herein, refers to the presence of a specified number of binding sites on an antigen-binding protein (such as a CAR or sdAb). For example, a native antibody or full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites on an antigen-binding protein (such as a CAR or sdAb), respectively.

[0065] "Chimeric antigen receptor" or "CAR," as used herein, refers to a genetically engineered receptor that can be used to graft one or more antigen specificities onto immune effector cells, such as T cells. Some CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T cell" refers to a T cell that expresses a CAR.

[0066] The terms "T cells" and "T lymphocytes" are interchangeable and used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+ CD8+ T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells. Also included are "NKT cells," which refer to a specialized population of T cells that express a semi-invariant αβ T cell receptor but also express various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8- cells. The TCR on NKT cells is unique in that it recognizes glycolipids presented by the MHC I-like molecule CD1d. NKT cells can have protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma-delta T cells (γδ T cells)," which refer to a specialized population of a small subset of T cells that possess a distinct TCR on their surface; unlike the majority of T cells, whose TCRs are composed of two glycoprotein chains, termed α- and β-TCR chains, the TCR in γδ T cells is composed of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immune regulation, are an important source of IL-17, and have been found to induce potent CD8+ cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which refer to T cells that suppress aberrant or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4+ T cells, but can also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0067] The term "siltacabtagene autoleucel" or "cilta-cel" refers to a chimeric antigen receptor T-cell (CAR-T) therapy containing two B-cell maturation antigen (BCMA)-targeting VHH domains designed to confer avidity for BCMA. Cilta-cel can comprise T lymphocytes transduced with the siltacabtagene autoleucel CAR, a CAR encoded by a retinoviral vector. The CAR targets a human B-cell maturation antigen (anti-BCMA CAR). A diagram of a retinoviral vector encoding the cilta-cel CAR is provided in Figure 2. The amino acid sequence of the cilta-cel CAR is that of SEQ ID NO: 17.

[0068] "Tumor cell" or "cancer cell" refers to a cancerous, precancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that undergoes spontaneous or induced phenotypic changes. These changes do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and the incorporation of new genomic nucleic acid, but it can also occur spontaneously or after exposure to carcinogens, resulting in the incorporation of exogenous nucleic acid, thereby mutating endogenous genes. Transformation / cancer is exemplified by morphological changes, cellular immortalization, aberrant growth control, lesion formation, proliferation, malignancy, modulation of tumor-specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo.

[0069] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have undergone natural or intervening modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can exist as a single chain or as two or more associated chains.

[0070] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides, and their analogs. "Oligonucleotide," as used herein, refers to short, generally single-stranded, synthetic polynucleotides that generally, although not necessarily, do not exceed about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure can include parent hybridoma cells as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition of nascent RNA transcripts from 5' to 3' is referred to as the transcription direction; the region of the sequence on the DNA strand that is 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence"; the region of the sequence on the DNA strand that is 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0071] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixture of nucleic acids, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding a single domain antibody or antibody as described herein are isolated or purified. This term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized in heterologous systems. A substantially pure molecule can include a molecule in isolated form. Specifically, an "isolated" nucleic acid molecule encoding a CAR or sdAb described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced.

[0072] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein, depending on the version, may contain one or more introns.

[0073] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0074] As used herein, the term "operably linked," and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refers to an operable linkage in which nucleic acid or amino acid sequences, respectively, are placed in a functional relationship with each other. For example, operably linking a promoter, enhancer element, open reading frame, 5' and 3' UTRs, and terminator sequence results in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in transcription of the open reading frame and ultimately production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one in which functional domains are positioned at an appropriate distance from each other to confer the intended function of each domain.

[0075] The term "vector" refers to a substance used to carry or contain nucleic acid sequences, including, for example, nucleic acid sequences encoding binding molecules (e.g., antibodies) as described herein, for introducing nucleic acid sequences into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selection sequences or markers that can serve for stable integration into a host cell chromosome. In addition, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included provide, for example, resistance to antibiotics or toxins, complement deficiencies of required nutrients, or supply important nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules (e.g., both antibody heavy and light chains or antibody VH and VL) are to be coexpressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acids can be operably linked to one common expression control sequence, or can be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods for testing expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules will be expressed in amounts sufficient to produce the desired product, and will further understand that expression levels can be optimized to achieve sufficient expression using methods well known in the art.

[0076] The term "host," as used herein, refers to an animal, such as a mammal (e.g., a human).

[0077] The term "host cell," as used herein, refers to the particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the host cell genome.

[0078] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0079] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0080] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0081] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency for use in animals, and more particularly in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0082] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0083] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0084] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation, suitable for use in contact with human and animal tissues or organs, without undue toxicity, irritation, allergic reaction, immunogenicity, or other problems or complications, and commensurate with a reasonable benefit-risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0085] In some embodiments, the excipient is a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc., including formulations.

[0086] Compositions, including pharmaceutical compounds, can contain a binding molecule (eg, an antibody), eg, in isolated or purified form, together with a suitable amount of excipients.

[0087] The term "effective amount" or "therapeutically effective amount," as used herein, refers to an amount of a single domain antibody or therapeutic molecule, including drugs and pharmaceutical compositions provided herein, sufficient to effect a desired result.

[0088] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, who has been diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, who is at risk of developing a disease or disorder.

[0089] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0090] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a decrease, alleviation, and / or mitigation of one or more symptoms associated with the underlying disorder, such that the patient experiences improvement even though the patient may still be suffering from the underlying disorder. The term "treatment" includes both maintenance and remission of the disease. The terms "maintain," "maintaining," and "maintenance" refer to the beneficial effect a subject derives from a therapy, but which does not necessarily result in a cure of the disease.

[0091] The term "line of treatment," when used in connection with the methods of treatment herein, refers to one or more cycles of a planned treatment program, which may consist of one or more planned cycles of monotherapy or combination therapy and a series of treatments administered in a planned manner. For example, a planned treatment regimen of induction therapy followed by autologous stem cell transplantation followed by maintenance is one line of treatment. A new line of treatment is considered to have been initiated when the planned course of treatment is modified to include another therapeutic agent or medication (alone or in combination) as a result of disease progression, recurrence, or toxicity. A new line of treatment is also considered to have been initiated when a planned period of observation-free treatment is interrupted by the need for additional treatment for the disease.

[0092] The term "resistant," when used in connection with treatment with a particular therapeutic agent or drug or line of therapy herein, refers to a disease or disease subject that fails to respond to said therapeutic agent or drug or line of therapy. The phrase "resistant myeloma" refers to multiple myeloma that is unresponsive during primary or salvage therapy or has progressed within 60 days of the last therapy.

[0093] The phrase "non-responsive disease" refers to either a failure to achieve a minimal response or the development of progressive disease during therapy.

[0094] "Enhance" or "promote" or "increase" or "expand" or "improve" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to the response caused by either a vehicle or a control molecule / composition. Measurable physiological responses can include, among other things, T cell expansion, activation, effector function, increased persistence, and / or increased cancer cell killing capacity, as understood in the art and as described herein. In certain embodiments, an "increased" or "enhanced" amount may be a "statistically significant" amount and may include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by a vehicle or control composition.

[0095] "Decrease" or "lower" or "reduce" or "reduce" or "alleviate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a physiological response (i.e., a downstream effect) that is smaller than the response caused by either a vehicle or a control molecule / composition. In certain embodiments, a "decreased" or "reduced" amount may be a "statistically significant" amount and may include a 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) decrease in the response caused by a vehicle, a control composition, or a response in a particular cell lineage (a reference response).

[0096] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of a disease, disorder, condition, or one or more associated symptoms (e.g., diabetes or cancer) occurring (or recurring).

[0097] As used herein, "delaying" the onset of cancer means extending, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease. This delay can be of varying lengths of time, depending on the course of the disease and / or individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that "delays" the onset of cancer is one that reduces the probability of disease development within a given time frame and / or reduces the extent of disease within a given time frame, compared to the absence of the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The onset of cancer may be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation studies, arteriography, or biopsy. Onset may also refer to the progression of cancer, which may be initially undetectable, and includes emergence, recurrence, and development.

[0098] "B-cell-related disease or disorder," as used herein, refers to a disease or disorder that is mediated by B cells or imparted by aberrant B-cell function (such as dysregulated B-cell function). "B-cell-related disease or disorder," as used herein, includes, but is not limited to, B-cell malignancies such as B-cell leukemia or B-cell lymphoma. It also includes marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal large B-cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia (B-PLL), follicular lymphoma (FL), Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B-cell lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), high-grade B-cell lymphoma (HGBL), and multiple myeloma (MM). "B-cell associated disease or disorder" also includes certain autoimmune and / or inflammatory diseases, such as those associated with inappropriate or enhanced B-cell numbers and / or activation.

[0099] "BCMA-associated disease or disorder," as used herein, refers to a disease or disorder involving cells or tissues in which BCMA is expressed or overexpressed. In some embodiments, a BCMA-associated disease or disorder involves cells on which BCMA is aberrantly expressed. In other embodiments, a BCMA-associated disease or disorder involves cells that are deficient in or on BCMA.

[0100] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0101] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0102] Whenever an embodiment is described herein with the term "comprising," it is understood that otherwise similar embodiments described with the term "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein with the phrase "consisting essentially of," it is also understood that otherwise similar embodiments described with the term "consisting of" are also provided.

[0103] The term "between," as used in phrases such as "between A and B" or "between A and B," refers to a range that includes both A and B.

[0104] Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be construed as specifically disclosing all the possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the range.

[0105] The term "and / or," as used in phrases such as "A and / or B," is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0106] 5.2. Methods for assessing response to CAR-T cell therapy In one aspect, provided herein are methods for assessing a subject's responsiveness to a therapy comprising T cells expressing a bivalent BCMA-targeting chimeric antigen receptor (CAR) based on the length of time the subject maintains a minimal residual disease (MRD)-negative state. In some embodiments, the method includes administering the T cells to the subject; measuring the length of time the subject maintains an MRD-negative state; and assessing the subject's responsiveness to the therapy based on the length of time the subject maintains an MRD-negative state.

[0107] Methods for determining MRD status and measuring the length of time a subject remains MRD-negative are known in the art, and any of these methods may be used in the present method.

[0108] In some embodiments, the method includes obtaining a bone marrow-derived sample (such as a bone marrow aspirate or biopsy) from the subject to assess MRD status. In certain embodiments, clinical staging (morphology, cytogenetics, and immunohistochemistry or immunofluorescence or flow cytometry) may be performed. In certain embodiments, a portion of the bone marrow aspirate may be immunophenotyped and monitored for BCMA, checkpoint ligand expression on CD138-positive multiple myeloma cells, and checkpoint expression on T cells. In certain embodiments, MRD may be monitored in the subject using next-generation sequencing (NGS) of bone marrow aspirate DNA. NGS of bone marrow aspirate DNA is known to those skilled in the art. In certain embodiments, NGS is performed via clonoSEQ. In certain embodiments, a baseline bone marrow aspirate may be used to define the myeloma clone, and a post-treatment sample may be used to assess MRD-negativity. In certain embodiments, MRD-negativity status may be based on evaluable samples. In certain embodiments, an evaluable sample has passed one or more, or all, of calibration, quality control, and sufficiency of evaluable cells at a particular sensitivity level. In some embodiments, the sensitivity level is about 10 -6In certain embodiments, the sensitivity level is about 10 -5 In certain embodiments, the sensitivity level is about 10 -4 In certain embodiments, the sensitivity level is about 10 -3 is.

[0109] In some embodiments, the methods comprise assessing the subject's responsiveness to the treatment based on MRD-negative status being less than 6 months. In some embodiments, the methods comprise assessing the subject's responsiveness to the treatment based on MRD-negative status being at least 6 months and less than 12 months. In other embodiments, the methods comprise assessing the subject's responsiveness to the treatment based on MRD-negative status being at least 12 months.

[0110] In some embodiments, the methods involve assessing the likelihood that the subject will have a complete response (CR), partial response (PR), stringent CR (sCR), or very good partial response (VGPR). In some embodiments, the methods involve determining that the subject is likely to have a CR, PR, sCR, or VGPR if MRD-negative status is maintained in the subject for less than six months. In some embodiments, the methods involve determining that the subject is likely to have a CR or sCR if MRD-negative status is maintained in the subject for more than six months.

[0111] In certain embodiments, a subject's response to treatment is assessed using response criteria based on the International Myeloma Working Group (IMWG), which are summarized in Table 6. In certain embodiments, a response may be classified as stringent complete response (sCR). In certain embodiments, a response may be classified as complete response (CR) that is worse than stringent complete response (sCR). In certain embodiments, a response may be classified as very good partial response (VGPR) that is worse than complete response (CR). In certain embodiments, a response may be classified as partial response (PR) that is worse than very good partial response (VGPR). In certain embodiments, a response may be classified as minimal response (MR) that is worse than partial response (PR). In certain embodiments, a response may be classified as stable disease (SD) that is worse than minimal response (MR). In certain embodiments, a response may be classified as progressive disease (PD) that is worse than stable disease.

[0112] In certain embodiments, the tests used to assess response criteria according to the International Myeloma Working Group (IMWG) are serum and urinary myeloma protein (M-protein) measurements, serum calcium corrected for albumin, bone marrow examination, skeletal examination, and documentation of extramedullary plasmacytoma.

[0113] Non-limiting examples of tests for measuring M-protein in blood and urine are known to those skilled in the art and include serum quantitative Ig, serum protein electrophoresis (SPEP), serum immunofixation electrophoresis, serum FLC assay, 24-hour urinary M-protein quantification by electrophoresis (UPEP), urine immunofixation electrophoresis, and serum β2-microglobulin.

[0114] Calculation of serum calcium corrected for albumin in a blood sample for the detection of hypercalcemia is known to those skilled in the art. Without wishing to be bound by theory, calcium binds to albumin, and only unbound (free) calcium is biologically active; therefore, serum calcium levels must be adjusted for abnormal albumin levels ("corrected serum calcium").

[0115] In certain embodiments, a skeletal survey of any one or all of the skull, entire spine, pelvis, thorax, humerus, femur, and any other bones may be performed, and both may be assessed by either radiography ("X-ray") or low-dose computed tomography (CT) diagnostic-quality scans without the use of IV contrast, as known to those skilled in the art. In certain embodiments, after T cell administration and before disease progression is confirmed, X-ray or CT scans may be performed locally whenever clinically indicated based on symptoms to document response or progression. In certain embodiments, magnetic resonance imaging (MRI) may be used to assess bone disease but does not replace a skeletal survey. MRI is known to those skilled in the art. In certain embodiments, if a radionuclide bone scan is used at screening, both methods may be used in addition to a complete skeletal survey to document disease status. Radionuclide bone scans are known to those skilled in the art. In certain embodiments, a radionuclide bone scan and a complete skeletal survey may be performed simultaneously. In certain embodiments, a radionuclide bone scan may not replace a complete skeletal survey. In certain embodiments, when a subject exhibits disease progression manifested by symptoms of pain due to bone changes, disease progression may be documented by skeletal survey or other radiographs, depending on the symptoms experienced by the subject.

[0116] In certain embodiments, extramedullary plasmacytoma may be documented by clinical examination or MRI. In certain embodiments, if there are no contraindications to the use of IV contrast, extramedullary plasmacytoma may be documented by CT scan. In certain embodiments, extramedullary plasmacytoma may be documented by fusion of positron emission tomography (PET) and CT scan if the CT component is of sufficient diagnostic quality. In certain embodiments, assessment of measurable areas of extramedullary disease may be performed, measured, or assessed locally in subjects every 4 weeks until the occurrence of confirmed CR or confirmed disease progression. In certain embodiments, assessment of extramedullary plasmacytoma may be done every 12 weeks.

[0117] In certain embodiments, to qualify for VGPR, PR, or MR, the sum of the products of perpendicular diameters of pre-existing extramedullary plasmacytomas may decrease by more than 90% or at least 50%, respectively. In certain embodiments, to qualify for disease progression, the sum of the products of perpendicular diameters of pre-existing extramedullary plasmacytomas must increase by at least 50%, or the longest diameter of a previous lesion with a short axis >1 cm must increase by at least 50%, or a new plasmacytoma must occur. In certain embodiments, to qualify for disease progression when all pre-existing extramedullary plasmacytomas have not been reported, the sum of the products of perpendicular diameters of reported plasmacytomas must have increased by at least 50%. In certain embodiments, if the test treatment interferes with the immunofixation assay, CR may be defined as the disappearance of the original M-protein associated with multiple myeloma on immunofixation.

[0118] In other embodiments, the method comprises, for example, assessing the likelihood of a subject having progression-free survival at a certain time point after treatment. In some embodiments, the method comprises determining that if the MRD-negative state is maintained in the subject for more than 6 months, the subject is likely to have progression-free survival for at least 12 months after treatment. In some embodiments, the method comprises determining that if the MRD-negative state is maintained in the subject for more than 12 months, the subject is likely to have progression-free survival for at least 24 months after treatment.

[0119] In yet other embodiments, the method includes assessing duration of response in the subject. In some embodiments, the method includes determining that if MRD-negative status is maintained in the subject for longer than 6 months, the subject is likely to have a duration of response greater than 12 months. In other embodiments, the method includes determining that if MRD-negative status is maintained in the subject for longer than 12 months, the subject is likely to have a duration of response greater than 24 months.

[0120] Bivalent BCMA-targeted CARs The T cells provided in the method express a bivalent BCMA-targeting CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a first VHH domain and a second VHH domain, wherein the first VHH domain comprises CDR1, CDR2, and CDR3 as set forth in the VHH domain comprising the amino acid sequence of SEQ ID NO: 2, and the second VHH domain comprises CDR1, CDR2, and CDR3 as set forth in the VHH domain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some specific embodiments, the first VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and the second VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first VHH domain comprises the amino acid sequence of SEQ ID NO: 2, and the second VHH domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first VHH domain is N-terminal to the second VHH domain. In other embodiments, the first VHH domain is C-terminal to the second VHH domain. In some embodiments, the first VHH domain is linked to the second VHH domain via a linker comprising the amino acid sequence of SEQ ID NO: 3.

[0121] The first and second VHH domains can be linked via a peptide linker. The different domains of the CAR can be fused to each other via peptide linkers.

[0122] Each peptide linker of a CAR can have the same or different length and / or sequence depending on the structural and / or functional characteristics of the antibody and / or various domains. Each peptide linker can be independently selected and optimized. The length, degree of flexibility, and / or other properties of one or more peptide linkers used in a CAR can have some effect on properties, including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. For example, a longer peptide linker can be selected to ensure that two adjacent domains do not sterically interfere with each other. In some embodiments, a short peptide linker can be placed between the transmembrane domain and the intracellular signaling domain of a CAR. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) to allow adjacent domains to move freely relative to each other. For example, a glycine-serine duo may be a suitable peptide linker.

[0123] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 amino acids in length or more. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids in length or less. In some embodiments, the length of the peptide linker is from about 1 amino acid to about 10 amino acids, from about 1 amino acid to about 20 amino acids, from about 1 amino acid to about 30 amino acids, from about 5 amino acids to about 15 amino acids, from about 10 amino acids to about 25 amino acids, from about 5 amino acids to about 30 amino acids, from about 10 amino acids to about 30 amino acids, from about 30 amino acids to about 50 amino acids, from about 50 amino acids to about 100 amino acids, or from about 1 amino acid to about 100 amino acids.

[0124] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Other linkers known in the art, for example, as described in International Publication No. WO 2016014789, International Publication No. WO 2015158671, International Publication No. WO 2016102965, US Patent Application Publication No. WO 20150299317, International Publication No. WO 2018067992, US Patent No. 7,741,465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference, can also be included in the CARs provided herein.

[0125] In some specific embodiments, the peptide linker connecting the first VHH domain and the second VHH domain comprises the amino acid sequence of SEQ ID NO:3.

[0126] The CAR of the present disclosure comprises a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain can be derived from either a natural source or a synthetic source. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain suitable for use in the CAR described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0127] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form an α-helix, a complex of two or more α-helices, a β-barrel, or any other stable structure capable of spanning a cellular phospholipid bilayer. Additionally, or alternatively, transmembrane domains can be classified based on their topology, including the number of passes the transmembrane domain makes across the membrane and the orientation of the protein. For example, a single-pass transmembrane protein crosses the cellular membrane once, while a multi-pass transmembrane protein crosses the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their termini relative to the inside and outside of the cell and the topology of one or more transmembrane segments. Type I membrane proteins have only one membrane-spanning segment and are oriented such that the N-terminus of the protein is on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins also have only one membrane-spanning segment, but are oriented so that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple membrane-spanning segments and can be further subdivided based on the number of transmembrane segments and the location of the N- and C-termini.

[0128] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, the transmembrane domain of a multi-pass membrane protein may also be compatible for use in the CAR described herein. A multi-pass membrane protein may comprise a complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helix or beta sheet structure. In some embodiments, the N- and C-termini of a multi-pass membrane protein are present on opposite sides of a lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.

[0129] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD2 78), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, Claudin-6, IL- 2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE , CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TN FR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(B Y55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0130] In some specific embodiments, the transmembrane domain is derived from CD8α, hi some embodiments, the transmembrane domain is the transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO:6.

[0131] The transmembrane domain used in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids, or more. Examples of synthetic transmembrane domains are known in the art, for example, in U.S. Pat. No. 7,052,906 and WO 2000 / 032776 (the relevant disclosures of which are incorporated herein by reference).

[0132] The transmembrane domains provided herein may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, which in some embodiments facilitates orientation of the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0133] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, i.e., hydrophobic or hydrophilic properties, of a protein or protein segment can be determined by any method known in the art, for example, Kyte and Doolittle hydropathy analysis.

[0134] The CAR of the present disclosure comprises an intracellular signaling domain. The intracellular signaling domain is involved in activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "cytoplasmic signaling domain" refers to a portion of a protein that transmits an effector function signal and directs a cell to perform a specialized function. Typically, the entire cytoplasmic signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain that is sufficient to transmit the effector function signal.

[0135] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that induces immune effector functions, playing a stimulatory role. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. As used herein, "ITAM" refers to a conserved protein motif typically present in the tails of signaling molecules expressed in many immune cells. This motif may contain two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids (where each x is independently any amino acid), resulting in the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated, at least in part, by phosphorylation of tyrosine residues in the ITAM upon activation of the signaling molecule. ITAMs may also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0136] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO:8.

[0137] Many immune effector cells require costimulation in addition to antigen-specific signal stimulation to promote cell proliferation, differentiation, and survival, and to activate cellular effector functions. In some embodiments, a CAR comprises at least one costimulatory signaling domain. The term "costimulatory signaling domain," as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response, such as an effector function. The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a costimulatory protein, which transmits signals and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

[0138] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., about any of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and the one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0139] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be compatible for use in the CARs described herein. The type or types of costimulatory signaling domains are selected based on factors such as the type of immune effector cell on which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of costimulatory signaling domains for use in CARs include, without limitation, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFFR / TNFRSF13C, and CTLA-4); , CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFRSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNFRII / TNFRSF1B);SLAM family members (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other costimulatory molecules, e.g., CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPR, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C;

[0140] In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of a ligand that specifically binds to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0141] In some embodiments, the intracellular signaling domain in a CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises the costimulatory signaling domain of CD137 comprising the amino acid sequence of SEQ ID NO:7.

[0142] Mutants of any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain has the ability to modulate the immune response of an immune cell, are also within the scope of the present disclosure. In some embodiments, the costimulatory signaling domain comprises up to 10 amino acid residue (e.g., 1, 2, 3, 4, 5, or 8) mutations when compared to its wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid mutations may be referred to as mutants. Mutating amino acid residues in a costimulatory signaling domain may result in increased signaling and enhanced stimulation of an immune response compared to a costimulatory signaling domain that does not contain the mutation. Mutating amino acid residues in a costimulatory signaling domain may result in decreased signaling and reduced stimulation of an immune response compared to a costimulatory signaling domain that does not contain the mutation.

[0143] The CAR of the present disclosure may comprise a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is generally an amino acid segment found between two domains of a protein, and may provide flexibility to the protein, allowing one or both domains to move relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule may be used.

[0144] The hinge domain can contain about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain can be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0145] In some embodiments, the hinge domain is the hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of the hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment comprising at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO:5.

[0146] Hinge domains of antibodies, such as IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is a hinge domain that connects the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of an antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0147] Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker such as a (GxS)n linker, where x and n can independently be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater.

[0148] CARs of the present disclosure may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide will target the effector molecule to the secretory pathway of a cell, allowing for incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences, that are compatible for use in the CARs described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of SEQ ID NO: 1.

[0149] In a specific embodiment, the CAR provided herein comprises the amino acid sequence of SEQ ID NO:17.

[0150] 5.4. Engineered Immune Effector Cells Immune effector cells (such as T cells) provided herein express a CAR, including those described herein (see Section 5.3, above). An "immune effector cell" is an immune cell capable of performing an immune effector function. In some embodiments, the immune effector cell expresses at least FcγRIII and performs ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0151] In some embodiments, the immune effector cells are T cells. In some embodiments, the T cells are CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or combinations thereof. In some embodiments, the T cells produce IL-2, TFN, and / or TNF when the CAR is expressed and bound to a target cell, such as a GPC3+ tumor cell. In some embodiments, the CD8+ T cells lyse antigen-specific target cells when the CAR is expressed and bound to the target cell.

[0152] The engineered immune effector cells are prepared by introducing a CAR into immune effector cells, such as T cells. In some embodiments, the CAR is introduced into the immune effector cells by transfecting them with any one of the isolated nucleic acids or vectors described above. In some embodiments, the CAR is introduced into the immune effector cells by inserting the protein into the cell membrane while passing the cells through a microfluidic system, such as CELL SQUEEZE® (see, e.g., U.S. Patent Application Publication No. 20140287509).

[0153] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical, or biological methods.

[0154] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, vectors are introduced into cells by electroporation.

[0155] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells, e.g., human cells.

[0156] Chemical means of introducing vectors into immune effector cells include macromolecule complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems, including oil-in-water emulsions, micelles, micellar mixtures, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro is a liposome (e.g., an artificial membrane vesicle).

[0157] In some embodiments, RNA molecules encoding any of the CARs described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into immune effector cells by known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035 (2006).

[0158] In some embodiments, the transduced or transfected immune effector cells are expanded ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are expanded in culture for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select modified mammalian cells.

[0159] Reporter genes can be used to identify potentially transfected cells and evaluate the function of regulatory sequences. Generally, reporter genes are genes that are not present or expressed in the recipient organism or tissue, and encode a polypeptide whose expression is manifested as some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0160] Other methods for confirming whether a nucleic acid encoding a CAR is present in an engineered immune effector cell include molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as detecting the presence or absence of specific peptides by immunological methods (such as ELISA and Western blot).

[0161] In some embodiments, a source of T cells is obtained from a subject prior to expansion and genetic modification of the T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, several T cell lines available in the art can be used. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using several techniques known to those skilled in the art, such as Ficoll™ separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution can be calcium-free and magnesium-free, or can be devoid of most, if not all, divalent cations. An initial activation step in the absence of calcium can lead to expanded activation. As one of ordinary skill in the art will readily appreciate, the washing step can be accomplished by methods known to those of ordinary skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processing machine, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be washed with, for example, Ca 2+ Contains no Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions with or without buffering agents. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0162] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated using positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In some embodiments, this time is approximately 30 minutes. In further embodiments, this time ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In further embodiments, this time is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, this incubation time is 10 to 24 hours. In some embodiments, the incubation time is 24 hours. For isolating T cells from leukemia patients, using a longer incubation time, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, in some embodiments, subpopulations of T cells can be preferentially selected for or culled out at the initiation of culture or at other time points during treatment by simply shortening or extending the time T cells are allowed to bind to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells. Additionally, subpopulations of T cells can be preferentially selected for or culled out at the initiation of culture or at other desired time points by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces. Those skilled in the art will recognize that multiple rounds of selection may also be used.In some embodiments, it may be desirable to perform a selection procedure and use the "unselected" cells for the activation and expansion process. The "unselected" cells can also be subjected to further rounds of selection.

[0163] Enrichment of T cell populations by negative selection can be achieved with a combination of antibodies against surface markers unique to the cells being negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are depleted using anti-CD25 conjugated beads or other similar selection methods.

[0164] For isolation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can vary. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum cell-to-bead contact. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml may be used. Using a higher concentration may result in increased cell yield, cell activation, and cell expansion. Furthermore, using a higher cell concentration may allow for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or cells from samples containing many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and may be desirable to obtain. In some embodiments, using a higher cell concentration allows for more efficient selection of CD8+ T cells, which typically have weak CD28 expression.

[0165] In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express high amounts of the desired antigen to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 than CD8+ T cells and are captured more efficiently at dilute concentrations. In some embodiments, the concentration of cells used is 5×10 6 In some embodiments, the concentration used is about 1 x 10 5 / mL ~ 1 × 10 6 / mL and any integer value in between.

[0166] In some embodiments, cells may be incubated on a rotator at either 2-10° C. or room temperature at various speeds for various lengths of time.

[0167] T cells for stimulation can also be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing step may result in a more uniform product due to the removal of granulocytes and some monocytes in the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and may be useful in this regard, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods, as well as uncontrolled immediate freezing at -20°C or into liquid nitrogen, can be used.

[0168] In some embodiments, cryopreserved cells are thawed, washed, and allowed to stand at room temperature for 1 hour before activation as described herein.

[0169] The present disclosure also contemplates collecting a blood sample or apheresis product from a subject at a time prior to when expanded cells as described herein may be needed. In this manner, a source of cells to expand can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk for developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and used at a later time. In certain embodiments, a sample is taken from a patient shortly after diagnosis of a particular disease as described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from a subject prior to any number of relevant therapeutic modalities, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Liu et al., Cell 66:807-815 (1991); Henderson et al., Immun 73:316-321 (1991); Bierer et al., Curr. Opin. Immun. 5:763-773 (1993)).In a further embodiment, cells are isolated for a patient and frozen for use after in conjunction with (e.g., before, concurrently with, or after) T cell ablative therapy using either bone marrow or stem cell transplant, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.

[0170] In some embodiments, T cells are obtained from a patient immediately after treatment. In this regard, it has been observed that with certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved in terms of their ex vivo expansion capacity during the period immediately following treatment, when the patient would normally be recovering from treatment. Similarly, with ex vivo manipulation using the methods described herein, the cells may be in a state favorable for enhanced engraftment and in vivo expansion. Thus, within the context of the present disclosure, it is contemplated to harvest blood cells, including T cells, dendritic cells, or other hematopoietic cells, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in a subject that favor the repopulation, recirculation, regeneration, and / or expansion of specific cell types, particularly during defined time windows following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0171] In some embodiments, prior to or after genetically modifying T cells with a CAR described herein, T cells may generally be engineered to express a CAR, such as those described in, e.g., U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7, T cells can be activated and expanded using methods such as those described in U.S. Patent Application Publication Nos. 067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0172] Generally, T cells can be expanded by contact with a surface having attached thereto an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, T cell populations can be stimulated as described herein, such as by contact with a surface-immobilized anti-CD3 antibody, or an antigen-binding fragment thereof, or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. Anti-CD3 antibody and anti-CD28 antibody can be used to stimulate proliferation of either CD4+ or CD8+ T cells. Examples of anti-CD3 antibodies include UCHT1, OKT3, and HIT3a (BioLegend, San Diego, USA), and may be used in the same manner as other methods commonly known in the art (Graves J, et al., J. Immunol. 146:2102 (1991); Li B, et al., Immunology 116:487 (2005); Rivollier A, et al., Blood 104:4029 (2004)). Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and may be used, as may other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977 (1998); Haanen et al., J. Exp. Med. 190(9):13191328 (1999); Garland et al., J. Immunol Meth. 227(1-2):53-63 (1999)).

[0173] In some embodiments, the primary stimulatory signal and the costimulatory signal to T cells can be provided by various protocols. For example, the agents providing each signal can be in solution or coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other conjugate that will bind to the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, for artificial antigen-presenting cells (aAPCs) contemplated for use in certain embodiments of the present disclosure for T cell activation and expansion.

[0174] In some embodiments, T cells are combined with drug-coated beads, followed by separation of the beads and cells, and then culturing the cells. In alternative embodiments, the drug-coated beads and cells are not separated but are cultured together prior to culturing. In further embodiments, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0175] By way of example, cell surface proteins can be ligated by contacting T cells with paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 4 ~4×10 8T cells) and beads (e.g., anti-CD3 / CD28 MACSiBead particles at a recommended titer of 1:100) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). One of skill in the art will readily appreciate that any cell concentration can be used. For example, target cells may be extremely rare in a sample, comprising as little as 0.01% of the sample, or the entire sample (i.e., 100%) may contain the desired target cells. Thus, any cell number is within the context of the present disclosure. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL are used. In yet another embodiment, cell concentrations of 75, 80, 85, 90, 95, or 100 million cells / mL are used. In further embodiments, concentrations of 125 or 150 million cells / mL may be used. Using higher concentrations may result in increased cell yield, cell activation, and cell expansion. Furthermore, using higher cell concentrations may enable more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and may be desirable to obtain in certain embodiments. For example, using higher cell concentrations may enable more efficient selection of CD8+ T cells, which typically have weak CD28 expression.

[0176] In some embodiments, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of one hour in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several stimulation cycles may be desirable, so that the T cell culture time can be 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for growing cells known to those of skill in the art. Other additives for growing cells include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or one or more cytokines in amounts sufficient for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental culture media and not in the culture media for cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to various stimulation times may exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product has a higher percentage of helper T cell populations (TH, CD4+) than cytotoxic or suppressor T cell populations (TC, CD8).Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors results in a T cell population that is primarily composed of TH cells before about day 8-9, while after about day 8-9, the T cell population becomes increasingly dominated by TC cells. Thus, depending on the therapeutic objective, it may be advantageous to infuse a T cell population that is primarily composed of TH cells into a subject. Similarly, if an antigen-specific TC cell subset is isolated, it may be beneficial to expand that subset to a greater extent.

[0177] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers vary widely, but most vary reproducibly over the course of the cell expansion process, thus enabling the ability to tailor activated T cell products to specific purposes.

[0178] In some embodiments, the method described herein uses 3.0×10 7 ~1.0×10 8 In some embodiments, a dosage form comprising 3.0 x 10 of the present CAR-T cells is provided. 7 ~1.0×10 8 Dosage forms comprising 3.0 x 10 of the present CAR-T cells are provided. In certain embodiments, the dosage form comprises 3.0 x 10 7 ~4.0×10 7 In a specific embodiment, the dosage form comprises 3.5 x 10 CAR-T cells. 7 ~4.5×10 7 In a specific embodiment, the dosage form comprises 4.0 x 10 CAR-T cells. 7 ~5.0×10 7 In a specific embodiment, the dosage form comprises 4.5 x 10 CAR-T cells. 7 ~5.5×10 7 In a specific embodiment, the dosage form comprises 5.0 x 10 CAR-T cells. 7 ~6.0×10 7 In a specific embodiment, the dosage form comprises 5.5 x 10 CAR-T cells. 7 ~6.5×10 7 In a specific embodiment, the dosage form comprises 6.0 x 10 CAR-T cells.7 ~7.0×10 7 In a specific embodiment, the dosage form comprises 6.5 x 10 CAR-T cells. 7 ~7.5×10 7 In a specific embodiment, the dosage form comprises 7.0 x 10 CAR-T cells. 7 ~8.0×10 7 In a specific embodiment, the dosage form comprises 7.5 x 10 CAR-T cells. 7 ~8.5×10 7 In a specific embodiment, the dosage form comprises 8.0 x 10 CAR-T cells. 7 ~9.0×10 7 In a specific embodiment, the dosage form comprises 8.5 x 10 CAR-T cells. 7 ~9.5×10 7 In a specific embodiment, the dosage form comprises 9.0 x 10 CAR-T cells. 7 ~1.0×10 8 Contains CAR-T cells.

[0179] In some embodiments, the cell population of the CAR-T dosage forms described herein includes, for example, T cells or populations of T cells at various stages of differentiation. Stages of T cell differentiation, from least differentiated to most differentiated, include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells. After antigen exposure, naive T cells proliferate and differentiate into memory T cells, e.g., stem central memory T cells and central memory T cells, which subsequently differentiate into effector memory T cells. Upon receiving appropriate T cell receptor, costimulatory, and inflammatory signals, memory T cells further differentiate into terminal effector T cells. See, e.g., Restifo. Blood. 124.4(2014):476-77; and Joshi et al. J. Immunol. 180.3(2008):1309-15.

[0180] Naive T cells may have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95-. Stem central memory T cells (Tscm) may have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T cells (Tcm) may have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (Tem) may have the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+. Terminal effector T cells (Teff) may have the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO-, CD95+. See, e.g., Gattinoni et al. Nat. Med. 17(2011):1290-7; and Flynn et al. Clin. Translat. Immunol. 3(2014):e20.

[0181] Vectors Polynucleotide sequences encoding the CARs described herein can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from antibody-producing cells, such as hybridoma cells, and sequenced. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques.

[0182] The present disclosure also provides a vector comprising a nucleic acid sequence encoding a CAR disclosed herein. The vector can be, for example, a plasmid, a cosmid, a viral vector (e.g., retroviral or adenoviral), or a phage. Suitable vectors and methods for vector preparation are well known in the art (see, for example, Sambrook et al. and Ausubel et al.).

[0183] In addition to the nucleic acid sequence encoding the CAR disclosed herein, the vector preferably contains expression control sequences, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc., that effect expression of the nucleic acid sequence in a host cell. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0184] In some embodiments, the vector comprises a promoter. Numerous promoters recognized by a variety of potential host cells are known. The selected promoter can be operably linked to the cistron DNA encoding the CAR disclosed herein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Numerous promoters, including constitutive, inducible, and repressible promoters from a variety of different sources, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences from repositories such as the ATCC and other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, CA), the LACSWITCH™ system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27:4324-4327 (1999); Nuc. Acid. Res., 28:e99 (2000); U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144 (2005)).

[0185] In some embodiments, the vector contains an "enhancer." The term "enhancer," as used herein, refers to, for example, a DNA sequence that increases transcription of an operably linked nucleic acid sequence. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of different sources are known in the art and are available as or within cloned polynucleotides (e.g., from repositories such as the ATCC and other commercial or private sources). Some polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of a coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within the immunoglobulin (Ig) locus. Such Ig enhancers include, for example, heavy chain (mu) 5' enhancers, light chain (kappa) 5' enhancers, kappa and mu intronic enhancers, and 3' enhancers (see generally Paul WE (ed.), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and U.S. Pat. No. 5,885,827).

[0186] In some embodiments, the vector comprises a "selection marker gene." The term "selection marker gene," as used herein, refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected for or against in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in WO 1992 / 08796 and WO 1994 / 28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981); Santerre et al., Gene, 30:147 (1984); Kent et al., Science, 237:901-903 (1987); Wigler et al. al., Cell, IP. 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026 (1962); Lowy et al., Cell, 22:817 (1980); and U.S. Pat. Nos. 5,122,464 and 5,770,359.

[0187] In some embodiments, a vector is an "episomal expression vector" or "episome," which is capable of replicating in a host cell and persists within the host cell as an extrachromosomal segment of DNA under appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pB-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use the SV40 origin of replication in place of T-antigen and EBNA1 and oriP.

[0188] In some embodiments, the vector is an "integrating expression vector," which may integrate randomly into the DNA of a host cell or may contain recombination sites that allow for recombination between the expression vector and specific sites in the chromosomal DNA of the host cell. Such integrating expression vectors may utilize endogenous expression control sequences of the host cell's chromosome to drive expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, CA) or components of the cre-lox system, such as can be found in the pExchange-6 Core vector from Stratagene (La Jolla, CA). Examples of vectors that integrate randomly into a host cell chromosome include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen (Carlsbad, CA) and pCI or pFNI OA(ACT)FLEXI™ from Promega (Madison, WI).

[0189] In some embodiments, the virus is a viral vector. Exemplary viral expression vectors include, but are not limited to, adenoviral vectors (e.g., the adenoviral Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentiviral vectors (e.g., the lentiviral pLP1 system from Life Technologies (Carlsbad, CA)), and retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, CA)). In a preferred embodiment, the viral vector is a lentiviral vector.

[0190] A vector containing a nucleic acid of the invention encoding a CAR can be introduced into a host cell capable of expressing the encoded CAR, including any suitable prokaryotic or eukaryotic cell. Preferred host cells are those that can be grown easily and reliably, have reasonably fast growth rates, have well-characterized expression systems, and can be easily and efficiently transformed or transfected.

[0191] As used herein, the term "host cell" refers to any type of cell that can contain an expression vector. Host cells can be eukaryotic cells, such as plants, animals, fungi, or algae, or prokaryotic cells, such as bacteria or protists. Host cells can be cultured or primary cells, i.e., isolated directly from an organism, e.g., a human. Host cells can be adherent or suspension cells, i.e., cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5α, Escherichia coli (E. coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. In a preferred embodiment, the host cell is a HEK293 cell. In some embodiments, the HEK293 cell is derived from the ATCC SD-3515 strain. In some embodiments, the HEK293 cell is derived from the IU-VPF MCB strain. In some embodiments, the HEK293 cell is derived from the IU-VPF MWCB strain. In some embodiments, the host cell may be a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC), or a natural killer (NK). Preferably, the host cell is a natural killer (NK) cell. More preferably, the host cell is a T cell.

[0192] To amplify or replicate a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. To produce a virus from a viral expression vector, the host cell can be a eukaryotic cell, such as a HEK293 cell. To produce a recombinant CAR, the host cell can be a mammalian cell. The host cell is preferably a human cell. The host cell can be of any cell type, derived from any tissue type, and at any developmental stage. Methods for selecting suitable mammalian host cells and for transforming, culturing, amplifying, screening, and purifying cells are known in the art.

[0193] In some embodiments, the present disclosure provides an isolated host cell that expresses a nucleic acid sequence encoding a CAR described herein.

[0194] In some embodiments, the host cell is a T cell. The T cells of the present disclosure can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, or T cells obtained from a mammal. When obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cells are preferably human T cells (e.g., isolated from a human). T cells can be at any developmental stage, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. In one embodiment, the T cells are CD8+ T cells or CD4+ T cells. T cell lines are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microorganisms and Cell Cultures (DSMZ), and include, for example, Jurkat cells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.

[0195] In some embodiments, the host cell is a natural killer (NK) cell. NK cells are a type of cytotoxic lymphocyte that plays a role in the innate immune system. NK cells are defined as large granular lymphocytes and constitute the third type of cell differentiated from a common lymphoid progenitor cell that also gives rise to B and T lymphocytes (see, e.g., Immunobiology, 5th ed., Janeway et al., eds., Garland Publishing, New York, NY (2001)). NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. After maturation, NK cells enter the circulation as large lymphocytes with characteristic cytotoxic granules. NK cells can recognize and kill some abnormal cells, such as some tumor cells and virus-infected cells, and are thought to be important in innate immune defense against intracellular pathogens. As described above with respect to T cells, the NK cells can be any NK cells, such as cultured NK cells, e.g., primary NK cells, or NK cells derived from cultured NK cells, or NK cells obtained from a mammal. If obtained from a mammal, the NK cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The NK cells can also be enriched or purified. The NK cells are preferably human NK cells (e.g., isolated from a human). NK cell lines are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), and derivatives thereof.

[0196] In some embodiments, a nucleic acid sequence encoding a CAR can be introduced into a cell by "transfection," "transformation," or "transduction." "Transfection," "transformation," or "transduction," as used herein, refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.

[0197] Many transfection techniques are known in the art, including calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-enhanced microprojectile bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after propagation of infectious particles in suitable packaging cells, many of which are commercially available.

[0198] 5.6. Pharmaceutical Compositions and Formulations Further provided herein is a pharmaceutical composition comprising any one of the anti-BCMA antibodies of the present disclosure or any one of the engineered immune effector cells comprising any one of the CARs (such as a BCMA CAR) as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by mixing any of the immune effector cells described herein having the desired purity in the form of a lyophilized formulation or aqueous solution with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). In certain embodiments, the pharmaceutical composition of CAR-T cells further comprises an excipient selected from dimethyl sulfoxide or dextran-40.

[0199] The compositions described herein can be administered as part of a pharmaceutical composition containing one or more carriers. The choice of carrier will be determined in part by the specific nucleic acid sequence, vector, or host cell expressing the CAR disclosed herein, as well as the specific method used to administer the nucleic acid sequence, vector, or host cell expressing the CAR disclosed herein. Thus, there are various suitable formulations of the pharmaceutical composition of the present disclosure.

[0200] For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives may optionally be used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0201] In addition, a buffering agent can be used in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents can be optionally used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[0202] Compositions comprising nucleic acid sequences encoding the CARs disclosed herein, or host cells expressing the CARs disclosed herein, can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes. Liposomes can serve to target host cells (e.g., T cells or NK cells) or the nucleic acid sequences disclosed herein to specific tissues. Liposomes can also be used to increase the half-life of the nucleic acid sequences disclosed herein. Many methods are available for preparing liposomes, such as those described in Szoka et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980), and U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369. The compositions can utilize time-release, delayed-release, and sustained-release delivery systems so that delivery of the compositions disclosed herein occurs before sensitization of the site to be treated and in sufficient time to cause sensitization. Many types of release delivery systems are available and known to those skilled in the art. Such systems can avoid repeated administration of the composition, thereby increasing convenience for the subject and the physician, and may be particularly suitable for certain composition embodiments of the present disclosure.

[0203] In certain embodiments, the CAR-T cells are about 1.0 x 10 5 ~2.0×10 5 cells / kg, 1.5×10 5 ~2.5×10 5 cells / kg, 2.0×10 5 ~3.0×10 5 cells / kg, 2.5×10 5 ~3.5×10 5 cells / kg, 3.0×10 5 ~4.0×10 5 cells / kg, 3.5×10 5 ~4.5×10 5 cells / kg, 4.0×10 5 ~5.0×10 5 cells / kg, 4.5×10 5 ~5.5×10 5cells / kg, 5.0×10 5 ~6.0×10 5 cells / kg, 5.5×10 5 ~6.5×10 5 cells / kg, 6.0×10 5 ~7.0×10 5 cells / kg, 6.5×10 5 ~7.5×10 5 cells / kg, 7.0×10 5 ~8.0×10 5 cells / kg, 7.5×10 5 ~8.5×10 5 cells / kg, 8.0×10 5 ~9.0×10 5 cells / kg, 8.5×10 5 ~9.5×10 5 cells / kg, or 9.0 x 10 5 ~1.0×10 6 In a preferred embodiment, the dose is about 0.75 x 10 cells / kg. 6 In certain embodiments, CAR-T cells are formulated at 1.0 x 10 cells / kg per subject. 8 It is formulated in subcellular doses.

[0204] 5.7. Methods of Treating Subjects The present disclosure further relates to methods and compositions for use in cellular immunotherapy. In some embodiments, the cellular immunotherapy is for treating cancer in a subject, including, but not limited to, hematological tumors and solid tumors. In some embodiments, the subject is a human. In some embodiments, the methods are suitable for treating adult and pediatric populations, including all age subsets, and may be used as any line of treatment, including first-line or subsequent lines.

[0205] The modified immune effector cells (such as CAR-T cells) described herein can be used in methods of treating cancer. In some embodiments, the immune effector cells are autologous. In some embodiments, the immune effector cells are allogeneic.

[0206] In certain embodiments, the CAR-T cells are about 1.0 x 10 5 ~2.0×10 5 cells / kg, 1.5×10 5 ~2.5×10 5 cells / kg, 2.0×10 5 ~3.0×10 5 cells / kg, 2.5×10 5 ~3.5×10 5 cells / kg, 3.0×10 5 ~4.0×10 5 cells / kg, 3.5×10 5 ~4.5×10 5 cells / kg, 4.0×10 5 ~5.0×10 5 cells / kg, 4.5×10 5 ~5.5×10 5 cells / kg, 5.0×10 5 ~6.0×10 5 cells / kg, 5.5×10 5 ~6.5×10 5 cells / kg, 6.0×10 5 ~7.0×10 5 cells / kg, 6.5×10 5 ~7.5×10 5 cells / kg, 7.0×10 5 ~8.0×10 5 cells / kg, 7.5×10 5 ~8.5×10 5 cells / kg, 8.0×10 5 ~9.0×10 5 cells / kg, 8.5×10 5 ~9.5×10 5 cells / kg, 9.0×10 5 ~1.0×10 6 cells / kg, 1.0×10 6 ~2.0×10 6 cells / kg, 1.5×10 6 ~2.5×10 6 cells / kg, 2.0×10 6 ~3.0×10 6 cells / kg, 2.5×10 6 ~3.5×10 6 cells / kg, 3.0×10 6 ~4.0×106 cells / kg, 3.5×10 6 ~4.5×10 6 cells / kg, 4.0×10 6 ~5.0×10 6 cells / kg, 4.5×10 6 ~5.5×10 6 cells / kg, or 5.0 x 10 6 ~6.0×10 6 In a preferred embodiment, the dose is about 0.75 x 10 cells / kg. 6 In certain embodiments, the CAR-T cells comprise about 1.0 x 10 cells / kg per subject. 8 The cells are administered in doses.

[0207] In certain embodiments, CAR-T cells are administered at a concentration of 1.0 x 10 per subject. 8 In certain embodiments, CAR-T cells are administered at a dose of about 3.0-4.0 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 3.5 to 4.5 x 10 7 In certain embodiments, CAR-T cells are administered at a dose of about 4.0-5.0 x 10 7 In certain embodiments, CAR-T cells are administered at a dose of about 4.5 to 5.5 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 5.0-6.0 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 5.5 to 6.5 x 10 cells. 7 In certain embodiments, the CAR-T cells are administered at a dose of about 6.0-7.0 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 6.5-7.5 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 7.0-8.0 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 7.5 to 8.5 x 10 7In certain embodiments, the CAR-T cells are administered at a dose of about 8.0-9.0 x 10 7 In certain embodiments, the CAR-T cells are administered at a dose of about 8.5-9.5 x 10 7 In a specific embodiment, the CAR-T cells are administered at a dose of about 9.0 x 10 7 ~1.0×10 8 The cells are administered in doses.

[0208] In certain embodiments, the CAR-T cells are about 0.693 x 10 6 In a specific embodiment, the CAR-T cells are administered at a dose of about 0.52 x 10 viable CAR-positive T cells / kg. 6 In certain embodiments, the CAR-T cells are administered at a dose of about 0.94 x 10 viable CAR-positive T cells / kg. 6 In a specific embodiment, the CAR-T cells are administered at a dose of about 0.709 x 10 CAR-positive viable T cells / kg. 6 In a specific embodiment, the CAR-T cells are administered at a dose of about 0.51 x 10 viable CAR-positive T cells / kg. 6 In certain embodiments, the CAR-T cells are administered at a dose of about 0.95 x 10 viable CAR-positive T cells / kg. 6 In certain embodiments, the CAR-T cells are administered in an outpatient setting.

[0209] In certain embodiments, CAR-T cells (e.g., any of the doses described above) are administered in one or more intravenous infusions. In certain embodiments, the administration of the CAR-T cells is via a single intravenous infusion. In certain embodiments, the single intravenous infusion is administered using a single bag of the CAR-T cells. In certain embodiments, the administration of the single bag of CAR-T cells is completed between the time the single bag of CAR-T cells is thawed and three hours after the single bag of CAR-T cells is thawed. In certain embodiments, a single intravenous administration is administered using two bags of the CAR-T cells. In certain embodiments, the administration of each of the two bags of CAR-T cells is completed between the time the first of the two bags of CAR-T cells is thawed and three hours after the first bag of CAR-T cells is thawed.

[0210] In certain embodiments, the time from initial apheresis to administration of CAR-T cells is less than 41, 47, 54, 61, 68, 75, 82, 89, 96, 103, 110, 117, 124, 131, 138, 145, 152, 159, 166, or 167 days. In certain embodiments, the time from initial apheresis to administration of CAR-T cells is greater than 41, 47, 54, 61, 68, 75, 82, 89, 96, 103, 110, 117, 124, 131, 138, 145, 152, 159, 166, or 167 days.

[0211] A composition comprising a host cell expressing a CAR-encoding nucleic acid sequence disclosed herein or a vector comprising a CAR-encoding nucleic acid sequence disclosed herein can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition is preferably suitable for parenteral administration. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Most preferably, the composition is administered by intravenous infusion.

[0212] A composition comprising a host cell expressing a CAR-encoding nucleic acid sequence disclosed herein, or a vector comprising a CAR-encoding nucleic acid sequence disclosed herein, can be administered to a mammal with one or more additional therapeutic agents, which can be co-administered. "Co-administration" refers to administering one or more additional therapeutic agents and a composition comprising a host cell disclosed herein or a vector disclosed herein sufficiently close in time that a CAR disclosed herein can potentiate the effect of the one or more additional therapeutic agents, or vice versa. In this regard, a composition comprising a host cell disclosed herein or a vector disclosed herein can be administered first, and one or more additional therapeutic agents can be administered second, or vice versa.

[0213] The CAR-expressing cells described herein and at least one additional therapeutic agent can be administered simultaneously in the same or separate compositions, or sequentially. For sequential administration, the CAR-expressing cells described herein can be administered first and the additional agent can be administered second, or the order of administration can be reversed.

[0214] In certain embodiments, the lymphocyte depletion regimen precedes administration of the CAR-T cells by about 5 to about 7 days. In certain embodiments, the lymphocyte depletion regimen is administered intravenously. In certain embodiments, the lymphocyte depletion regimen includes administration of cyclophosphamide or administration of fludarabine. In certain embodiments, the cyclophosphamide is administered at a dose of 300 mg / m 2 In certain embodiments, the fludarabine is administered intravenously at a dose of 30 mg / m 2 In certain embodiments, the dose is administered intravenously at 300 mg / m 2 Cyclophosphamide administered intravenously at 30 mg / m 2 A lymphocyte depletion regimen including fludarabine administered intravenously at RT precedes administration of the CAR-T cells by about 5 to about 7 days.

[0215] In certain embodiments, the subject further undergoes a bridging therapy, the bridging therapy comprising short-term treatment with at least one bridging therapy between apheresis and the lymphocyte-depleting regimen, wherein the at least one bridging therapy previously resulted in an outcome of stable disease, minimal response, partial response, best partial response, complete response, or stringent complete response for the subject. In certain embodiments, the subject has had an increase in tumor burden despite the bridging therapy. In certain embodiments, the subject has had an increase in tumor burden of about 25% or more despite the bridging therapy.

[0216] In certain embodiments, the subject is treated with a premedication regimen comprising an antipyretic and an antihistamine up to about 1 hour prior to the administration of the CAR-T cells. In certain embodiments, the antipyretic comprises either paracetamol or acetaminophen. In certain embodiments, the antipyretic is administered to the subject either orally or intravenously. In certain embodiments, the antipyretic is administered to the subject at a dose between 650 mg and 1000 mg. In certain embodiments, the antihistamine comprises diphenhydramine. In certain embodiments, the antihistamine is administered to the subject either orally or intravenously. In certain embodiments, the antihistamine is administered at a dose between 25 mg and 50 mg, or its equivalent. In certain embodiments, the antipyretic comprises either paracetamol or acetaminophen, and the antipyretic comprises a dose between 650 mg and 1000 mg administered to the subject either orally or intravenously, and the antihistamine comprises diphenhydramine, and the antihistamine comprises a dose between 25 mg and 50 mg, or the equivalent thereof, administered to the subject either orally or intravenously.

[0217] In some embodiments, the method further comprises diagnosing the subject for cytokine release syndrome (CRS). In a preferred embodiment, the diagnosis is made according to the consensus grading classification of the American Society of Transplantation and Cellular Therapy (ASTCT), formerly the American Society for Blood and Marrow Transplantation (ASBMT). A non-limiting summary of the ASTCT consensus grading classification for CRS diagnosis is provided in Table 7. In some embodiments, CRS is assessed by assessing levels of one or more, or all, of IL-6, IL-10, IFN-γ, C-reactive protein (CRP), and ferritin.

[0218] In some embodiments, the method further comprises treating the subject for cytokine release syndrome (CRS). In some embodiments, the treatment for CRS is with an antipyretic. In some examples, the treatment for CRS is with anticytokine therapy. In some embodiments, the treatment for CRS occurs more than about 3 days after infusion. In some embodiments, the treatment for CRS occurs without significantly reducing CAR-T cell expansion in vivo. In certain embodiments, the method further comprises treating the subject for cytokine release syndrome more than about 3 days after the administration of the CAR-T cells without significantly reducing the expansion of the CAR-T cells in vivo. In some embodiments, the treatment for CRS comprises administering to the subject an IL-6R inhibitor. In some embodiments, the IL-6R inhibitor is an antibody. In some embodiments, the antibody inhibits IL-6R by binding to its extracellular domain. In some embodiments, the IL-6R inhibitor prevents IL-6 from binding to IL-6R. In some embodiments, the IL-6R inhibitor is tocilizumab. In some embodiments, the anti-cytokine therapy comprises administration of tocilizumab. In some embodiments, the anti-cytokine therapy comprises administration of a steroid. In some embodiments, the treatment of CRS comprises treatment with a monoclonal antibody other than tocilizumab. In some embodiments, the antibody other than tocilizumab targets a cytokine. In some embodiments, the cytokine targeted by the antibody other than tocilizumab is IL-1. In some embodiments, the IL-1 targeting antibody is anakinra. In some embodiments, the cytokine targeted by the antibody other than tocilizumab is TNFα. In some embodiments, the treatment of CRS comprises administering a corticosteroid to the subject. In some embodiments, the treatment of CRS comprises use of a vasopressor. In some embodiments, the treatment of CRS comprises intubation or mechanical ventilation. In some embodiments, the treatment of CRS comprises administering cyclophosphamide to the subject. In some embodiments, the treatment of CRS comprises administering etanercept to the subject. In some embodiments, the treatment of CRS comprises administering levetiracetam to the subject. In some embodiments, treatment of CRS includes supportive care.

[0219] In some embodiments, the method further comprises diagnosing the subject for immune cell effector-associated neurotoxicity (ICANS). In some embodiments, the diagnosis is made according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) criteria. In some embodiments, the diagnosis is made according to the NCI CTCAE criteria, version 5.0. In some embodiments, the diagnosis is made according to the American Society of Transplantation and Cellular Therapy (ASTCT) consensus grading system. In some embodiments, neurotoxicity consistent with ICAN is present. A non-limiting summary of the ASTCT consensus grading system for ICANS diagnosis is provided in Table 8. In some embodiments, treating ICANS comprises administering to the subject an IL-6R inhibitor. In some embodiments, the IL-6R inhibitor is an antibody. In some embodiments, the antibody inhibits IL-6R by binding to its extracellular domain. In some embodiments, the IL-6R inhibitor prevents IL-6 from binding to IL-6R. In some embodiments, the IL-6R inhibitor is tocilizumab. In some embodiments, the treatment of ICANS comprises administering to the subject an IL-1 inhibitor. In some embodiments, the IL-1 inhibitor is an antibody. In a preferred embodiment, the IL-1 inhibitor antibody is anakinra. In some embodiments, the treatment of ICANS comprises administering to the subject a corticosteroid. In some embodiments, the treatment of ICANS comprises administering to the subject levetiracetam. In some embodiments, the treatment of ICANS comprises administering to the subject dexamethasone. In some embodiments, the treatment of ICANS comprises administering to the subject methylprednisone sodium succinate. In some embodiments, the treatment of ICANS comprises administering to the subject pethidine.In some embodiments, treatment of ICANS comprises administering to the subject one or more, or all, of tocilizumab, anakinra, corticosteroids, levetiracetam, dexamethasone, methylprednisone sodium succinate, or pethidine.

[0220] In some embodiments, the method further comprises diagnosing the subject for cytopenia. In some embodiments, the cytopenia comprises one or more, or all, of lymphopenia, neutropenia, and thrombocytopenia. Without being bound by theory, lymphopenia of Grade 3 or Grade 4, but not Grade 2 or less, is characterized by a lymphopenia of 0.5 x 10 per liter of blood sample from the subject. 9Grade 3 or 4 neutropenia, but not Grade 2 or less, is characterized by a neutrophil count of less than 1000 cells per microliter of the subject's blood sample, and Grade 3 or 4 thrombocytopenia, but not Grade 2 or less, is characterized by a platelet cell count of less than 50,000 cells per microliter of the subject's blood sample. In some embodiments, more than 75% of subjects with Grade 3 or 4 lymphopenia after administration of the CAR-T cells recover to Grade 2 or less lymphopenia 60 days after administration of the CAR-T cells. In some embodiments, more than 80% of subjects with Grade 3 or 4 lymphopenia after administration of the CAR-T cells recover to Grade 2 or less lymphopenia 60 days after administration of the CAR-T cells. In some embodiments, more than 85% of subjects with Grade 3 or 4 lymphopenia after administration of the CAR-T cells recover to Grade 2 or less lymphopenia 60 days after administration of the CAR-T cells. In some embodiments, greater than 90% of subjects with grade 3 or grade 4 lymphopenia after administration of the CAR-T cells recover to lymphopenia of grade 2 or less 60 days after administration of the CAR-T cells. In some embodiments, greater than 70% of subjects with grade 3 or grade 4 neutropenia after administration of the CAR-T cells recover to neutropenia of grade 2 or less 60 days after administration of the CAR-T cells. In some embodiments, greater than 75% of subjects with grade 3 or grade 4 neutropenia after administration of the CAR-T cells recover to neutropenia of grade 2 or less 60 days after administration of the CAR-T cells. In some embodiments, greater than 80% of subjects with grade 3 or grade 4 neutropenia after administration of the CAR-T cells recover to neutropenia of grade 2 or less 60 days after administration of the CAR-T cells. In some embodiments, greater than 85% of subjects with grade 3 or grade 4 neutropenia after CAR-T cell administration recover to grade 2 or less neutropenia 60 days after CAR-T cell administration.In some embodiments, more than 30% of subjects with grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recover to grade 2 or less thrombocytopenia 60 days after administration of the CAR-T cells. In some embodiments, more than 34% of subjects with grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recover to grade 2 or less thrombocytopenia 60 days after administration of the CAR-T cells. In some embodiments, more than 38% of subjects with grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recover to grade 2 or less thrombocytopenia 60 days after administration of the CAR-T cells. In some embodiments, more than 42% of subjects with grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recover to grade 2 or less thrombocytopenia 60 days after administration of the CAR-T cells.

[0221] When a host cell expressing a CAR-encoding nucleic acid sequence disclosed herein or a composition comprising a vector containing a CAR-encoding nucleic acid sequence disclosed herein is administered to a mammal (e.g., a human), the biological activity of the CAR can be measured by any suitable method known in the art. According to the methods disclosed herein, the CAR binds to BCMA on multiple myeloma cells, and the multiple myeloma cells are destroyed. The binding of the CAR to BCMA on the surface of multiple myeloma cells can be assayed using any suitable method known in the art, including, for example, ELISA and flow cytometry. The ability of the CAR to destroy multiple myeloma cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). The biological activity of a CAR can also be measured by assaying the expression of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF.

[0222] The methods described herein can be used to treat a variety of cancers, including both solid and liquid cancers. In certain embodiments, the methods are used to treat multiple myeloma. The methods described herein can be used as a first, second, third, or combination therapy in an adjuvant or neoadjuvant setting, along with other types of cancer therapy known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, and radiofrequency ablation.

[0223] In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is stage I, stage II, or stage III, and / or stage A or stage B multiple myeloma according to the Dury-Salmon staging system. In certain embodiments, the cancer is stage I, stage II, or stage III multiple myeloma according to the International Staging System published by the International Myeloma Working Group (IMWG). In some embodiments, the multiple myeloma agent is aggressive.

[0224] In certain embodiments, the subject has received at least one prior line of treatment. In certain embodiments, at least one prior line of treatment includes treatment with a medication that is a proteasome inhibitor (PI). Non-limiting examples of PIs include bortezomib, carfilzomib, and ixazomib. In certain embodiments, at least one prior line of treatment includes treatment with a medication that is an immunomodulatory drug (IMiD). Non-limiting examples of IMiDs include lenalidomide, pomalidomide, and thalidomide. In certain embodiments, at least one prior line of treatment includes treatment with a medication that is a corticosteroid. Non-limiting examples of corticosteroids include dexamethasone and prednisone. In certain embodiments, at least one prior line of treatment includes treatment with a medication that is an alkylating agent. In certain embodiments, at least one prior line of treatment includes treatment with a medication that is an anthracycline. In certain embodiments, at least one prior line of treatment includes treatment with a medication that is an anti-CD38 antibody. Non-limiting examples of anti-CD38 antibodies include daratumumab, isatuximab, and the investigational antibody TAK-079. In certain embodiments, at least one prior line of treatment includes treatment with a pharmaceutical agent that is elotuzumab. In certain embodiments, at least one prior line of treatment includes treatment with a pharmaceutical agent that is panobinostat. In certain embodiments, the subject has relapsed after said at least one prior line of treatment. In certain embodiments, the cancer is resistant to one or more, or all of, bortezomib, carfilzomib, ixazomib, lenalidomide, pomalidomide, thalidomide, dexamethasone, prednisone, alkylating agents, daratumumab, isatuximab, TAK-079, elotuzumab, and panobinostat. In certain embodiments, the prior line of treatment includes surgery, radiation therapy, or autologous or allogeneic transplantation, or any combination of such treatments.

[0225] In some embodiments, the multiple myeloma is resistant to at least two medications. In some embodiments, the multiple myeloma is resistant to at least three medications. In some embodiments, the multiple myeloma is resistant to at least four medications. In some embodiments, the multiple myeloma is resistant to at least five medications.

[0226] In some embodiments, the subject has between about 10% and about 30% bone marrow plasma cells prior to said administration of said CAR-T cells.

[0227] Methods of Treating Lenalidomide-Resistant Subjects

[0228] In one aspect, methods of treating a subject are provided, the method comprising administering to the subject a composition comprising a therapeutically effective number of T cells comprising a chimeric antigen receptor (CAR), wherein the subject has multiple myeloma and is lenalidomide-resistant. In some embodiments, the subject has received one, two, or three prior lines of therapy.

[0229] In some embodiments, the multiple myeloma is refractory to the last line of therapy. In some embodiments, the subject has relapsed after one, two, or three prior lines of therapy. In some embodiments, the subject has received at least one prior line of therapy comprising treatment with lenalidomide and at least one non-lenalidomide medication, wherein the at least one non-lenalidomide medication comprises at least one of a proteasome inhibitor, an immunomodulatory agent, or an anti-CD38 antibody. In some embodiments, the subject has no prior exposure to a BCMA-targeted medication. In some embodiments, the subject has received at least two prior lines of therapy. In some embodiments, the subject has received three prior lines of therapy.

[0230] In some embodiments, the subject has received prior treatment with dexamethasone, an alkylating agent, or daratumumab. In some embodiments, the multiple myeloma is resistant to three classes of medications.

[0231] Methods of Treating a Subject with a Prior Early Relapse

[0232]

[0010] In one aspect, a method of treating a subject is provided, the method comprising administering to the subject a composition comprising a therapeutically effective number of T cells comprising a chimeric antigen receptor (CAR), wherein the subject has multiple myeloma and has had a prior early relapse. The term "prior early relapse" means disease progression according to International Myeloma Working Group (IMWG) response criteria either (i) between the time of treatment with autologous stem cell transplantation (ASCT) and about 12 months after said treatment with autologous stem cell transplantation (ASCT) for participants who have had an autologous stem cell transplant (ASCT); or (ii) between the initiation of anti-myeloma therapy and about 12 months after initiation of anti-myeloma therapy for participants who have not had an autologous stem cell transplant (ASCT).

[0233] In some embodiments, the subject has received prior treatment with one prior line of therapy. In some embodiments, the one prior line of therapy comprises treatment with at least two medications. In some embodiments, the at least two medications comprise a proteasome inhibitor and an immunomodulatory agent. In some embodiments, the subject has been further treated with an anti-CD38 antibody. In some embodiments, the subject has had no prior exposure to a BCMA-targeted medication. In some embodiments, the multiple myeloma is resistant to at least one medication.

[0234] Methods of treating a subject with a non-cellular BCMA-targeted conditioning therapy

[0235]

[0010] In one aspect, provided are methods of treating a subject, the method comprising administering to the subject a composition comprising a therapeutically effective number of T cells comprising a chimeric antigen receptor (CAR), wherein the subject has multiple myeloma and has received at least one prior line of therapy comprising treatment with a non-cellular BCMA-targeted medication. In some embodiments, the at least one prior line of therapy comprises treatment with at least four medications, wherein the at least four medications comprise a non-cellular BCMA-targeted medication. In some embodiments, the at least four medications further comprise a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody.

[0236] In some embodiments, the subject has received prior treatment with at least two prior lines of treatment, at least three prior lines of treatment, at least four prior lines of treatment, at least five prior lines of treatment, at least six prior lines of treatment, at least seven prior lines of treatment, at least eight prior lines of treatment, at least nine prior lines of treatment, at least ten prior lines of treatment, at least eleven prior lines of treatment, or at least twelve prior lines of treatment. In some embodiments, the subject has relapsed after said at least one prior line of treatment, at least two prior lines of treatment, at least three prior lines of treatment, at least four prior lines of treatment, at least five prior lines of treatment, at least six prior lines of treatment, at least seven prior lines of treatment, at least eight prior lines of treatment, at least nine prior lines of treatment, at least ten prior lines of treatment, at least eleven prior lines of treatment, or at least 12 prior lines of treatment.

[0237] 5.8. Manufacturing kits and articles Any of the compositions described herein can be included in a kit. In some embodiments, the modified immortalized CAR-T cells are provided in the kit, which can also include reagents suitable for expanding the cells, such as media.

[0238] In non-limiting examples, a chimeric receptor expression construct, one or more reagents for making the chimeric receptor expression construct, cells for transfection of the expression construct, and / or one or more tools for obtaining immortalized T cells for transfection of the expression construct (such tools can be syringes, pipettes, tweezers, and / or any such medically approved instruments).

[0239] In some aspects, the kit includes reagents or equipment for electroporation of cells.

[0240] In some embodiments, the kit comprises an artificial antigen-presenting cell.

[0241] Kits may include one or more suitably aliquoted compositions of the present disclosure or reagents for making compositions of the present disclosure. Kit components may be packaged in either aqueous media or lyophilized form. The container means of the kit may include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be disposed, preferably suitably aliquoted. Where more than one component is present in the kit, the kit will also generally contain second, third, or other additional containers into which the additional components may be separately placed. However, various combinations of components may be contained in vials. Kits of the present disclosure will also typically include a means for containing the chimeric receptor construct and any other reagent containers in close confinement for commercial sale. Such containers may include, for example, injection-molded or blow-molded plastic containers into which the desired vials are retained.

[0242] For the sake of brevity, certain abbreviations are used herein. An example is the one-letter abbreviation for amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: [Table 2]

[0243] The present disclosure is generally disclosed herein using affirmative language in describing many embodiments. The present disclosure also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays, or analyses, is completely or partially excluded. Thus, even if the present disclosure does not generally explicitly state what it does not include, aspects not expressly included in the present disclosure are nevertheless disclosed herein.

[0244] Although several embodiments of the present disclosure have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, the following examples are intended to illustrate, but not limit, the scope of the disclosure as set forth in the claims. [Example]

[0245] The following is a description of various methods and materials used in this work, presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their disclosure, nor is it intended to represent that the experiments that follow are all that have been performed or could be performed. It should be understood that exemplary descriptions written in the present tense have not necessarily been performed, but rather that such descriptions could be performed to generate data or the like relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be accounted for.

[0246] 6.1. Example 1 - Siltakabtagene Autoreucel B-cell maturation antigen (BCMA), also known as CD269 and TNFRSF17, is a 20-kilodalton, type III membrane protein that is part of the tumor necrosis receptor superfamily. BCMA is a cell surface antigen expressed at high levels predominantly on B-lineage cells. Figure 1 shows the expression of BCMA on various immune-derived cells. Comparative studies have demonstrated its absence in most normal tissues and its lack of expression on CD34-positive hematopoietic stem cells. BCMA binds to two ligands that induce B-cell proliferation and plays an important role in B-cell maturation and subsequent differentiation into plasma cells. Its selective expression and biological significance for the proliferation and survival of myeloma cells make BCMA a promising target for CAR-T-based immunotherapy, siltacabtagene autoreucel.

[0247] Siltacabbutagen autoleucel is an autologous chimeric antigen receptor T cell (CAR-T) therapy that targets BCMA. The siltacabbutagen autoleucel chimeric antigen receptor (CAR) contains two B cell maturation antigen (BCMA)-targeting VHH domains designed to confer avidity. A map of the construct is shown in Figure 2. Siltacabbutagen autoleucel contains a VHH domain comprising the amino acid sequence set forth in SEQ ID NO:2 and a VHH domain comprising the amino acid sequence set forth in SEQ ID NO:4.

[0248] 6.2. Example 2. Method of Treatment with Siltacabtagene Autoleucel (Cilta-cel) Herein, we describe a Phase 1b-2, open-label, multicenter study we conducted to evaluate the safety and efficacy of cilta-cel butagene autoleucel in adult subjects with relapsed or refractory multiple myeloma. In the Phase 1b portion of the study, the recommended Phase 2 dose (RP2D) of cilta-cel was confirmed. In Phase 2, subjects were treated at the RP2D established from Phase 1b. The objective of the Phase 2 portion of the study was to further establish the safety and efficacy of cilta-cel. A schematic overview of the study flow chart, consisting of lymphocyte-depleting regimens prior to cilta-cel infusion, is shown in Figure 3.

[0249] The first analysis was conducted approximately 6 months after the last subject received their first dose of cilta-cel. This report is generated from the first analysis specified in the protocol. A summary of subjects enrolled in the study is shown in Table 1, and percentages were calculated using the number of subjects in all enrolled analyses set as the denominator. A total of 113 subjects (Phase 1b: 35; Phase 2: 78) were enrolled (apheresed) in the United States, of which 101 subjects (Phase 1b: 30; Phase 2: 71) received the conditioning regimen and 97 subjects (Phase 1b: 29; Phase 2: 68) received the cilta-cel infusion, which they received at the targeted RP2D. These 97 subjects comprised the all-treatment analysis set, which is the basis for all efficacy and safety analyses presented below. At the clinical cutoff, the median follow-up period based on Kaplan-Meier product limit estimation for the all-treated analysis set was 12.4 months. A summary of the study follow-up period is shown in Table 2, which lists the follow-up period relative to the date of the first cilta-cel infusion (Day 1).

[0250] The patient population was screened to include those with relapsed or refractory multiple myeloma and three prior lines or dual resistance to a PI / IMiD and previous PI, IMiD, and anti-CD38 exposure, where the PI is a proteasome inhibitor and the IMiD is an immunomodulatory agent. Another possible medication is an alkylating agent (ALKY). A summary of prior treatments received by the study subjects is shown in Table 3, and a summary of the resistance status of our study subjects to prior multiple myeloma treatments is shown in Table 4. Eligible patients were 18 years of age or older and had a diagnosis of MM according to the International Myeloma Working Group (IMWG) diagnostic criteria, measurable disease at baseline, and an Eastern Cooperative Oncology Group (ECOG) performance status score of 0, 1, or 2. The demographic and disease characteristics of the patient population in the phase 1b portion of the study are shown in Figure 4.

[0251] Eligible subjects underwent apheresis for peripheral blood mononuclear cell (PBMC) collection. Study enrollment was defined on the day of apheresis. Siltacabtagene Autoleucel formulation (DP) was produced from T cells selected from the apheresis. Subjects whose apheresis or production failed were allowed a second attempt at apheresis.

[0252] Bridging therapy (anti-plasma cell-specific treatment between apheresis and the first dose of the conditioning regimen) was permitted if clinically necessary (i.e., to maintain disease stability while waiting for siltacabtagene autoreucel manufacturing). Additional cycles of bridging therapy were considered based on the subject's clinical status and the timing of CAR-T product availability. Bridging therapy is defined as short-term treatment that previously produced at least a stable disease response in the subject.

[0253] After meeting the safety criteria for treatment, subjects received a conditioning regimen to help achieve lymphodepletion in the subject and promote CAR-T cell expansion. The lymphodepletion regimen consisted of cyclophosphamide 300 mg / m daily for 3 days. 2 and fludarabine 30 mg / m 2 The study included intravenous (IV) administration of cyclophosphamide 300 mg / m before the cilta-cel infusion. 2 and fludarabine 30 mg / m 2 This is consistent with the lymphocyte-depleting regimens used in the marketed CAR-T products Kymriah and Yescarta.

[0254] Five to seven days after the initiation of the conditioning regimen, cilta-cel prepared from apheresis material via viral transduction as shown in Figure 5 was administered on the day defined as day 1. Approximately 1 hour before cilta-cel infusion, subjects received premedication. Corticosteroids were not used during the pre-infusion. Pre-infusion medications are listed in Table 5. Following treatment with pre-infusion medications, cilta-cel administration was 1.0 x 108 with a maximum total dose of 0.75 x 10 CAR-positive viable T cells 6 CAR-positive viable T cells / kg (range: 0.5–1.0 × 10 6 The total targeted dose of 1000 CAR-positive viable T cells / kg was administered in a single infusion.

[0255] The silta-cbutagen autoleucel dose was contained in one or two cryopreserved patient-specific infusion bags. The timing of cilta-cel thawing was coordinated with the timing of the infusion. The infusion time was confirmed in advance, and the start of thawing was timed so that cilta-cel would be available for infusion when the patient was ready. If more than one bag was received for a therapeutic infusion, one bag was thawed at a time. The thawing / infusion of the next bag was delayed until the previous bag was deemed safe to administer.

[0256] The post-infusion period began after completion of the cilta-cel infusion on day 1 and continued until day 100. The post-treatment period began on day 101 and continued until study completion, defined as 2 years after the last subject received their first dose of cilta-cel. The development and persistence of cilta-cel, as measured by blood concentrations, are summarized in Figure 6.

[0257] 6.3. Example 3. Evaluation of the effectiveness of the method of treatment with Siltacabtagene Autoreucel Using the IMWG-based response criteria summarized in Table 6, this study classified responses, from better to worse, as either stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR), minimal response (MR), stable disease, or progressive disease. Disease progression was recorded consistently across clinical trial sites. The studies performed to evaluate the IMWG-based response criteria are as follows:

[0258] Myeloma protein measurements in serum and urine: Myeloma protein (M-protein) measurements were made from blood and 24-hour urine samples using the following tests: serum quantitative Ig, serum protein electrophoresis (SPEP), serum immunofixation electrophoresis, serum FLC assay (for subjects in suspected CR / sCR and at all disease evaluations for subjects with serum FLC-only disease), 24-hour urinary M-protein quantification by electrophoresis (UPEP), urine immunofixation electrophoresis, and serum β2-microglobulin. Disease progression based on only one of the laboratory tests was confirmed by at least one repeat investigation. Disease evaluation continued beyond relapse from CR until disease progression was confirmed. Serum and urine immunofixation and serum free light chain (FLC) assays were performed at screening and thereafter if CR was suspected (when serum or 24-hour urine M-protein electrophoresis [by SPEP or UPEP] was 0 or not quantifiable). For subjects with light-chain multiple myeloma, serum and urine immunofixation tests were performed periodically.

[0259] Serum calcium corrected for albumin: Blood samples for calculating serum calcium corrected for albumin were collected and analyzed until the onset of confirmed disease progression; the occurrence of hypercalcemia (corrected serum calcium >11.5 mg / dL [>2.9 mmol / L]), if not attributable to any other cause, may indicate disease progression or recurrence. Calcium is bound to albumin, and only unbound (free) calcium is biologically active; therefore, serum calcium levels must be adjusted for abnormal albumin levels ("corrected serum calcium").

[0260] Bone marrow examination: Bone marrow aspirates or biopsies were performed for clinical evaluation. Bone marrow aspirates were performed for biomarker evaluation. Clinical staging (morphology, cytogenetics, and immunohistochemistry or immunofluorescence or flow cytometry) was performed. A portion of the bone marrow aspirate was immunophenotyped and monitored for checkpoint ligand expression on BCMA, CD138-positive multiple myeloma cells, and checkpoint expression on T cells. When feasible, bone marrow aspirates were also performed to confirm CR and sCR and at the time of disease progression. Furthermore, because minimal residual disease (MRD) negativity was evaluated as a potential surrogate for PFS and OS in multiple myeloma treatment, MRD was monitored in subjects using next-generation sequencing (NGS) on bone marrow aspirate DNA. Baseline bone marrow aspirates were used to define the myeloma clone, and post-treatment samples were used to evaluate MRD negativity. Fresh bone marrow aspirates were collected before (≤7 days) the first dose of the conditioning regimen.

[0261] Skeletal Examination: A skeletal examination (including the skull, entire spine, pelvis, thorax, humerus, femur, and any other bones the investigator suspects may be complicated by disease) was performed during the screening phase and was assessed by either radiography ("X-ray") or low-dose computed tomography (CT) scan without the use of IV contrast. If a CT scan was used, it was of diagnostic quality. After cilta-cel infusion and before disease progression was confirmed, X-rays or CT scans were performed locally whenever clinically indicated based on symptoms to document response or progression. Magnetic resonance imaging (MRI) was an accepted method for assessment of bone disease and was optionally included; however, it did not replace the skeletal examination. If a radionuclide bone scan was used at screening, both methods were used to document disease status in addition to the complete skeletal examination. These tests were performed simultaneously. The radionuclide bone scan did not replace the complete skeletal examination. If a subject showed disease progression manifested by symptoms of pain due to bone changes, disease progression was documented by skeletal survey or other radiographs, depending on the symptoms experienced by the subject. If the diagnosis of disease progression was clear by radiographic investigation, it was not considered necessary to perform a repeat confirmatory x-ray. If changes were equivocal, a repeat x-ray was performed in 1 to 3 weeks.

[0262] Documenting extramedullary plasmacytoma: The site of known extramedullary plasmacytoma was documented ≤14 days before the first dose of the conditioning regimen. Clinical examination or MRI was used to document the extramedullary location of disease. CT scan evaluation was considered an acceptable option if there was no contraindication to the use of IV contrast. Positron emission tomography scans or ultrasound studies were not acceptable for documenting the size of extramedullary plasmacytomas. However, PET / CT fusion scans were optionally used to document extramedullary plasmacytomas if the CT component of the PET / CT fusion scan was not of sufficient diagnostic quality. Extramedullary plasmacytomas were assessed by clinical examination or radiological imaging ≤14 days before the first dose of the conditioning regimen for all subjects with a history of plasmacytoma or if clinically indicated. Evaluation of measurable areas of extramedullary disease was performed, measured, and assessed locally every 4 weeks (for physical examination) for subjects with a history of plasmacytoma, or as clinically indicated during treatment for other subjects, until the occurrence of a confirmed CR or confirmed disease progression. If evaluation could only be performed radiologically, evaluation of extramedullary plasmacytoma was performed every 12 weeks. Irradiated or resected lesions were considered nonmeasurable and were monitored only for disease progression. To qualify for VGPR or PR / minimal response (MR), the sum of the products of perpendicular diameters of existing extramedullary plasmacytomas must have decreased by more than 90% or at least 50%, respectively, and no new plasmacytomas could have developed. To qualify for disease progression, the sum of the products of perpendicular diameters of existing extramedullary plasmacytomas must have increased by at least 50%, or the longest diameter of a previous lesion with a short axis >1 cm must have increased by at least 50%, or a new plasmacytoma must have developed. Although not all preexisting extramedullary plasmacytomas were reported, the criterion for disease progression was met if the sum of the products of the perpendicular diameters of the reported plasmacytomas increased by at least 50%.

[0263] If the study treatment was determined to have interfered with the immunofixation assay, CR was defined as the disappearance of the original M-protein associated with multiple myeloma in the immunofixation, and the determination of CR was not influenced by unrelated M-protein secondary to the study treatment.

[0264] Study endpoints, as assessed by an Independent Review Committee (IRC), were:

[0265] MRD was assessed in patients at baseline, day 28, and at 6, 12, 18, and 24 months of follow-up using next-generation sequencing (clonoSEQ version 2.0) (Adaptive Biotechnologies, Seattle, WA, USA) at the time of suspected complete response, and subsequently every 12 months until disease progression for patients who remained on study. MRD-negative patients passed calibration or quality control and were classified as 10 -5 Durability of MRD-negative status was assessed by estimating MRD-negativity rates at 6- and 12-month follow-up.

[0266] Clinical benefit rate (CBR) was defined as the proportion of subjects who achieved MR or better according to the IMWG criteria (sCR+CR+VGPR+PR+MR).

[0267] Overall response rate (ORR) was defined as the proportion of subjects achieving PR or better according to the IMWG criteria (sCR+CR+VGPR+PR).

[0268] VGPR or better response rate was defined as the proportion of subjects achieving a VGPR or better response according to the IMWG criteria (sCR+CR+VGPR).

[0269] Duration of response (DOR) was calculated among responders (PR or better response) from the date of first documented response (PR or better) as defined by IMWG criteria to the date of first documented evidence of progressive disease. Relapse from CR with positive immunofixation or trace amounts of M-protein was not considered disease progression. Disease assessment continued past relapse from CR until disease progression was confirmed.

[0270] Time to response (TTR) was defined as the time between the date of initial cilta-cel infusion and the first efficacy assessment in which the subject met all criteria for PR or better.

[0271] Progression-free survival (PFS) was defined as the time from the first infusion of cilta-cel to the date of first documented disease progression, or to the date of death from any cause, whichever occurred first, as defined by the IMWG criteria.

[0272] Overall survival (OS) was measured from the date of the first infusion of cilta-cel to the date of subject death.

[0273] For ORR, the response rate and its 95% exact confidence interval (CI) were calculated based on a binomial distribution, and the null hypothesis was rejected if the lower limit of the CI exceeded 30%. Analyses of VGPR or better response rate, DOR, PFS, and OS were performed using the same cutoffs as for ORR. Survival efficacy endpoints (DOR, PFS, and OS) were estimated using the Kaplan-Meier method. The distribution of DOR (median and Kaplan-Meier curve) was provided using Kaplan-Meier estimates. Similar analyses were performed for OS, PFS, and TTR.

[0274] 6.4. Example 4. Evaluation of the safety of the treatment regimen with Siltacabtagene Autoreucel Adverse events, except for CRS and CAR-T cell-related neurotoxicity (e.g., ICANS), were tracked, reported, and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE version 5.0). CRS was assessed according to the ASTCT consensus grading system, summarized in Table 7. At the first sign of CRS (e.g., fever), subjects were immediately admitted to the hospital for evaluation. Tocilizumab intervention was optionally used to treat subjects who showed symptoms of fever when other sources of fever were eliminated. Tocilizumab was optionally used for early treatment in subjects with a high risk of severe CRS (e.g., high baseline tumor burden, early onset of fever, or persistent fever 24 hours after symptomatic treatment). Other monoclonal antibodies targeting cytokines (e.g., anti-ILI and / or anti-TNFα) were optionally used, particularly for cases of CRS that did not respond to tocilizumab.

[0275] CAR-T cell-associated neurotoxicity (e.g., ICANS) was graded using the ASTCT consensus grading system summarized in Table 8. In addition, all individual symptoms of CRS (e.g., fever, hypotension) and ICANS (e.g., decreased level of consciousness, seizures) were captured as individual adverse events and graded according to the CTCAE criteria. Neurotoxicity that was not temporally associated with CRS or any other neurological adverse event that did not qualify as ICANS was graded according to the CTCAE criteria. Any adverse event or serious adverse event not listed in the NCI CTCAE version 5.0 was graded according to the investigator's clinical judgment by using standard grades as follows:

[0276] Grade 1: Mild: Asymptomatic or mildly symptomatic; clinical or diagnostic observations only; no need for intervention indicated.

[0277] Grade 2: Moderate; minimal, local or non-invasive intervention indicated; age restriction - adequate instrumental activities of daily living.

[0278] Grade 3: Severe or medically significant but not immediately life-threatening; indicates need for hospitalization or prolonged hospitalization; disabling; limiting self-care activities of daily living.

[0279] Grade 4: Life-threatening consequences; indicates the need for urgent intervention.

[0280] Grade 5: Death related to an adverse event.

[0281] Based on independent review committee (IRC) assessment, the response and duration of response for responders in the all-treated analysis set was analyzed at a median follow-up period of 12.4 months. The best overall response for subjects in the all-treated analysis set is summarized in Table 9. In the all-treated analysis set, 94 subjects (96.9%) achieved a PR or better response, and 65 subjects (67.0%) achieved a complete response (CR) or better, based on IRC assessment, resulting in a CBR of 96.9%. The deep and durable response induced by siltacabtagene autoleucel was demonstrated by a 92.8% VGPR or better rate and a 67.0% CR or better rate, and a median DOR not reached at the clinical cutoff with a median follow-up period of 12.4 months. The metrics used to evaluate the efficacy of siltacabtagene autoleucel are summarized below.

[0282] Tumor burden reduction: Tumor burden was reduced in 100% of subjects.

[0283] Overall response rate (ORR): 96.9% of subjects had an overall response with 95% exact CI (91.2%, 99.4%).

[0284] VGPR or better: 90 subjects (92.8% of subjects) achieved VGPR (very good partial response) or better.

[0285] Duration of response (DOR): The median DOR was not reached at months with a 95% CI of (15.9, NE); the probability of a responder remaining in response at 9 and 12 months was 80.2% (95% CI: 70.4%, 87.0%) and 68.2% (95% CI: 54.4%, 78.6%), respectively. The DOR for all responders in the all-treated analysis set is summarized in Table 10.

[0286] Time to response (TTR): The median time to first response (PR or better) and median time to best response were 0.95 and 2.56 months, respectively.

[0287] Progression-free survival (PFS): Median PFS was not reached in months with a 95% CI of 16.79 (NE); 9-month and 12-month PFS rates (95% CI) were 80.3% (70.9%, 87.0%) and 76.6% (66.0%, 84.3%), respectively. A summary of PFS in the all-treated analysis set is shown in Table 11.

[0288] Overall survival (OS): 14 subjects (14.4%) died at the time of clinical cutoff, with 9-month and 12-month overall survival rates (95% CI) of 90.7% (82.8%, 95.0%) and 88.5% (80.2%, 93.5%), respectively. OS based on the all-treated analysis set is summarized in Table 12.

[0289] Minimal residual disease (MRD) negativity rate (10 -5 The MRD-negative rate was 54.6% (95% CI: 44.2%, 64.8%), and 33 (34.0%) subjects achieved MRD-negative CR / sCR. -5 A summary of the overall MRD negativity rate in Table 13 for all subjects in the all-treated analysis set and in Table 14 for all subjects in the all-treated analysis set. -5 The thresholds are shown for subjects with evaluable samples at 100% confidence intervals (CI). Evaluable samples were those that passed calibration and quality control and contained sufficient cells for evaluation at each test threshold.

[0290] Siltacabtagene autoreucel was determined to have a safety profile consistent with the mechanism of action of CAR-T therapy.

[0291] CRS: CAR-T cell-related adverse events of CRS were common (94.8%) but mostly low-grade. CRS of all grades was reported in 92 (94.8%) subjects, as assessed by the ASTCT consensus grading system. All events of CRS resolved, except for one (1.1%) fatal event from a subject with a 97-day duration of CRS. A summary of treatment-emergent CRS events in the all-treated analysis set is shown in Table 15.

[0292] Immune effector cell-associated neurotoxicity (ICANS): ICANS of all grades, as assessed by the ASTCT consensus grading system, were reported in 16 (16.5%) subjects. All events resolved. A summary of ICANS with onset after cilta-cel infusion in the all-treated analysis set is shown in Table 16.

[0293] Cytopenias: Grade 3 or 4 cytopenias, including lymphopenia, neutropenia, and thrombocytopenia, were common in the post-infusion period; however, the majority of these events resolved by Day 60. 96 (99.0%), 95 (97.9%), and 60 (61.9%) subjects had Grade 3 or 4 lymphopenia, neutropenia, and thrombocytopenia, respectively, after the first 100 days of cilta-cel infusion. 88 (90.7%), 85 (87.6%), and 41 (42.3%) subjects had their initial Grade 3 or 4 events resolved to Grade 2 or less by Day 60 for lymphopenia, neutropenia, and thrombocytopenia, respectively. A summary of cytopenias after treatment with cilta-cel in the all-treated analysis set is shown in Table 17.

[0294] In conclusion, siltacabtagene autoleucel, as a single agent and single infusion, demonstrated unprecedented clinical efficacy in a heavily pretreated patient population, including an ORR of 96.9% and rapid onset of response in less than 1 month.

[0295] 6.5. Example 5. Efficacy Outcomes and Characteristics of Patients with Multiple Myeloma (MM) Who Achieved Sustained Minimal Residual Disease Negativity After Treatment with Siltacabtagene Autoleucel (cilta-cel) Cilta-cel is a BCMA-targeted CAR T-cell therapy recently approved by the US FDA for the treatment of adult patients (pts) with relapsed / refractory multiple myeloma (RRMM) after ≥4 prior lines of therapy (LOT), including proteasome inhibitors (PIs), immunomodulatory agents (IMiDs), and anti-CD38 monoclonal antibodies. In the phase 1b / 2 CARTITUDE-1 trial, cilta-cel demonstrated deep and durable responses in heavily pretreated pts with RRMM. The ORR was 97.9%, and no DOR was reached at a median follow-up of 28 months (mo). 61 of 97 pts were evaluable for minimal residual disease (MRD) negativity (clonoSEQ v2.0, Adaptive Biotechnologies), and 56 of these 61 pts (91.8%) remained MRD-negative (≥10%) at any time point. -5 ) was achieved. MRD negativity was maintained for <6 months in 22 pts, 6-12 months in 10 pts, and ≥12 months in 24 pts. This analysis sought to characterize baseline and disease characteristics of pts with persistent MRD negativity (pts who remained MRD negative for ≥6 and ≥12 months).

[0296] Eligible pts had MM, had received ≥3 prior therapies or were refractory to PIs and IMiDs, and received a PI, IMiD, and anti-CD38 antibody. Pts received a single cilta-cel infusion (target dose 0.75 × 10) 5-7 days after lymphodepletion. 6Patients received 10 CAR+ viable T cells / kg. MRD negativity was a secondary objective of the phase 2 portion of CARTITUDE-1 and was assessed for bone marrow samples using next-generation sequencing at baseline; day 28; and months 6, 12, 18, and 24, regardless of disease status. Additional samples were collected and evaluated at the time of suspected complete response and every 12 months until progressive disease (PD) for pts who remained on study. Evaluable samples passed calibration and quality control and were evaluated for 10 -5 Sufficient cells were included for evaluation at the study threshold. Characteristics were analyzed in pts with MRD negativity for <6 months or persistent ≥6 and ≥12 months. Pts who did not achieve MRD negativity at any time point were considered MRD positive. Landmark analyses were performed at 6 and 12 months to address immortality time bias.

[0297] Among the 56 pts who achieved MRD negativity in CARTITUDE-1, 50 had at least 6 months of follow-up without progression after initial MRD negativity, and 44 had at least 12 months of follow-up without progression after initial MRD negativity. MRD negativity was maintained for ≥6 months in 68% (34 of 50 with at least 6 months of follow-up without progression after initial MRD negativity) and ≥12 months in 55% (24 of 44 with at least 12 months of follow-up without progression after initial MRD negativity; all 6 pts evaluable for persistent MRD of ≥6 months but not ≥12 months had PD). Pts with persistent MRD negativity (≥6 months and ≥12 months) had longer progression-free survival (PFS; Figures 7 and 12) compared with pts who did not (MRD negativity <6 months; consistent with results from landmark analysis). Duration of response in MRD subgroups is shown in Figure 8. Responders with sustained MRD negativity had a longer DOR compared with those who did not. Key pt and disease characteristics in these different groups (Figure 9), as well as potential associations with sustained MRD negativity, were analyzed descriptively. Baseline extramedullary plasmacytoma was less common in pts with maintained MRD for ≥6 months (8.8%, n=3 / 34) and ≥12 months (4.2%, n=1 / 24) compared with pts with MRD negativity for <6 months (18.2%, n=4 / 22). All pts with sustained MRD negativity for ≥6 months achieved a stringent complete response (sCR) (Figure 11). Pts with sustained MRD negativity also had a trend toward a longer median time since diagnosis (5.9 and 7.0 years for pts with MRD negativity sustained ≥6 and 12 months, respectively, compared with 4.8 years for those with MRD negativity <6 months). Overall, pts who achieved sustained MRD negativity for ≥6 months had a deeper response than those with MRD negativity maintained for <6 months.Other baseline characteristics, including the presence of high-risk cytogenetics, ECOG performance status, ISS stage, number of prior LOTs, penta-drug resistance, and resistance to the last LOT, did not differ between MRD subgroups and were similar to the overall CARTITUDE-1 population.

[0298] Based on our descriptive analysis, pts receiving cilta-cel achieved MRD negativity despite their high-risk cytogenetics and ISS status, number of prior lot-of-treatment (LOT), and penta-drug resistance. While MRD negativity alone (<6 months) did not appear to offer a PFS benefit compared with MRD-positive pts, the small number of pts in the MRD-positive group (Figure 10) precludes comparison with those who achieved MRD negativity. In general, patients who did not achieve MRD negativity at any time point (MRD-positive) responded to cilta-cel. The presence of extramedullary plasmacytoma at baseline and time since diagnosis may be factors influencing the achievement of sustained MRD negativity. These data suggest that while cilta-cel is effective in a wide range of pts, certain pts and disease characteristics may be associated with sustained MRD negativity and favorable long-term outcomes.

[0299] From the foregoing, it will be understood that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope provided herein. All of the references referenced above are incorporated herein by reference in their entirety. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] array SEQ ID NO:1 - Siltakabtagene Autoreucel CAR CD8α signal peptide, CD8α SP amino acid sequence MALPVTALLLPLALLLHAARP SEQ ID NO:2 - Siltakabutagen Autoleucel CAR BCMA binding domain, VHH1 amino acid sequence [ka] SEQ ID NO:3 - Siltakabutagen Autoleucel CAR BCMA binding domain, G4S linker amino acid sequence GGGGS SEQ ID NO:4 - Siltakabutagen Autoleucel CAR BCMA binding domain, VHH2 amino acid sequence [ka] SEQ ID NO:5 - Siltakabtagene Autoreucel CAR CD8α hinge amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO:6 - Siltakabutagen Autoleucel CAR CD8α transmembrane amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO:7 - Siltakabtagene Autoreucel CAR CD137 cytoplasmic amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO:8 - Siltakabtagene Autoreucel CAR CD3ζ cytoplasmic amino acid sequence [ka] SEQ ID NO:9 - Siltakabtagene Autoreucel CAR CD8α signal peptide CD8α SP nucleic acid sequence ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCTGCTCGCCCT SEQ ID NO:10 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH1 nucleic acid sequence [ka] SEQ ID NO:11 - Siltakabtagene Autoleucel CAR BCMA binding domain, G4S linker nucleic acid sequence GGAGGAGGAGGATCT SEQ ID NO: 12 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH2 nucleic acid sequence [ka] SEQ ID NO:13 - Siltakabtagene Autoreucel CAR CD8α hinge nucleic acid sequence [ka] SEQ ID NO: 14 - Siltakabtagene Autoleucel CAR CD8α transmembrane nucleic acid sequence ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC SEQ ID NO: 15 - Siltakabtagene Autoreucel CAR CD137 cytoplasmic nucleic acid sequence [ka] SEQ ID NO: 16 - Siltakabtagene Autoreucel CAR CD3ζ cytoplasmic nucleic acid sequence [ka] SEQ ID NO: 17 - Siltakabutagen Autoreucel CAR amino acid sequence [ka] SEQ ID NO: 18 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH1 CDR1 SHVMG SEQ ID NO: 19 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH1 CDR2 VIGWRDISTSYADSVKG SEQ ID NO:20 - Siltakabutagen Autoleucel CAR BCMA binding domain, VHH1 CDR3 ARRIDAADFDS SEQ ID NO:21 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH2 CDR1 TFTMG SEQ ID NO:22 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH2 CDR2 AISLSPTLAYYAESVKG SEQ ID NO:23 - Siltakabtagene Autoleucel CAR BCMA binding domain, VHH2 CDR3 ADRKSVMSIRPDY

Claims

1. 1. A method for assessing a subject's responsiveness to a therapy comprising T cells expressing a bivalent BCMA-targeting chimeric antigen receptor (CAR), comprising: (a) administering to the subject the T cells; (b) determining the length of time the subject remains minimal residual disease (MRD) negative; and (c) (i) the MRD-negative status has been maintained for less than six months; (ii) the MRD-negative status has been maintained for at least 6 months and less than 12 months; or (iii) assessing the subject's responsiveness to the treatment based on maintenance of the MRD-negative status for at least 12 months; 2. A method according to claim 1, wherein the bivalent BCMA-targeting CAR comprises an extracellular antigen-binding domain comprising a first VHH domain and a second VHH domain, a transmembrane domain, and an intracellular signaling domain, wherein the first VHH domain comprises CDR1, CDR2, and CDR3 as set forth in the VHH domain comprising the amino acid sequence of SEQ ID NO: 2, and the second VHH domain comprises CDR1, CDR2, and CDR3 as set forth in the VHH domain comprising the amino acid sequence of SEQ ID NO:

4.

2. The method of claim 1, wherein the first VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 18, CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and the second VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO:

23.

3. The method of claim 1, wherein the first VHH domain comprises the amino acid sequence of SEQ ID NO: 2 and the second VHH domain comprises the amino acid sequence of SEQ ID NO:

4.

4. The method of any one of claims 1 to 3, wherein the first VHH domain is N-terminal to the second VHH domain, or the first VHH domain is C-terminal to the second VHH domain.

5. The method of any one of claims 1 to 4, wherein the first VHH domain is linked to the second VHH domain via a linker comprising the amino acid sequence of SEQ ID NO:

3.

6. 6. The method of any one of claims 1 to 5, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

7. The method of claim 6, wherein the transmembrane domain is derived from CD8α and comprises the amino acid sequence of SEQ ID NO:

6.

8. 8. The method of any one of claims 1 to 7, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

9. 9. The method of claim 8, wherein the primary intracellular signaling domain is derived from CD3ζ comprising the amino acid sequence of SEQ ID NO:

8.

10. 10. The method of any one of claims 1 to 9, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

11. 11. The method of claim 10, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and any combination thereof.

12. The method of claim 11, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD137 comprising the amino acid sequence of SEQ ID NO:

7.

13. The method of any one of claims 1 to 12, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

14. The method of claim 13, wherein the hinge domain is derived from CD8α comprising the amino acid sequence of SEQ ID NO:

5.

15. The method of any one of claims 1 to 14, wherein the CAR further comprises a signal peptide located at the N-terminus of the polypeptide.

16. 16. The method of claim 15, wherein the signal peptide is derived from CD8α comprising the amino acid sequence of SEQ ID NO:

1.

17. The method of any one of claims 1 to 16, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

17.

18. The method of any one of claims 1 to 17, wherein the subject has a disease or disorder.

19. 19. The method of claim 18, wherein the disease or disorder is cancer.

20. 20. The method of claim 19, wherein the cancer is multiple myeloma.

21. 21. The method of claim 20, wherein the cancer is resistant or relapsed multiple myeloma.

22. 22. The method of any one of claims 1 to 21, wherein the method comprises obtaining a bone marrow aspirate or biopsy from the subject to assess MRD status.

23. 23. The method of any one of claims 1 to 22, wherein the MRD status is monitored using next generation sequencing (NGS) of bone marrow aspirate DNA.

24. 24. The method of claim 23, wherein the NGS is performed via clonoSEQ.

25. 25. The method of any one of claims 22-24, wherein a baseline bone marrow aspirate is used to define the myeloma clone and a post-treatment sample is used to assess MRD negativity.

26. 26. The method of any one of claims 22 to 25, wherein the evaluable sample has passed one or more, or all, of calibration, quality control, and sufficiency of evaluable cells at a particular sensitivity level.

27. The sensitivity level is about 10 －６ , 10 －５ , 10 －４ , or 10 －３ 27. The method of claim 26, wherein:

28. 28. The method of any one of claims 1-27, wherein the length of time the subject maintains an MRD-negative state is measured from the time MRD is first achieved in the subject.

29. 29. The method of any one of claims 1-28, wherein the method comprises assessing the likelihood that the subject will have a complete response (CR), a partial response (PR), a stringent CR (sCR), or a very good PR (VGPR).

30. 30. The method of claim 29, comprising determining that the subject is likely to have a CR, PR, sCR, or VGPR if the MRD-negative status is maintained in the subject for less than 6 months.

31. 30. The method of claim 29, comprising determining that the subject is likely to have a CR or sCR if the MRD-negative status is maintained in the subject for greater than 6 months.

32. The method of any one of claims 1 to 28, wherein said method comprises assessing the subject's likelihood of having progression-free survival.

33. 33. The method of claim 32, comprising determining that if the MRD-negative status is maintained in the subject for longer than 6 months, the subject is likely to have progression-free survival for at least 12 months after the treatment.

34. 33. The method of claim 32, comprising determining that if the MRD-negative status is maintained in the subject for greater than 12 months, the subject is likely to have progression-free survival for at least 24 months following the treatment.

35. The method of any one of claims 1 to 28, wherein the method comprises assessing duration of response in the subject.

36. 36. The method of claim 35, comprising determining that the subject is likely to have a duration of response greater than 12 months if the MRD-negative status is maintained in the subject for longer than 6 months.

37. 37. The method of claim 36, comprising determining that the subject is likely to have a duration of response greater than 24 months if the MRD-negative status is maintained in the subject for greater than 12 months.

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