Methods of treating smoldering multiple myeloma with ciltacabtagene autoleucel

Abstract

Provided herein are methods of treating smoldering multiple myeloma in a subject in need thereof. In some embodiments, the method comprises administering ciltacabtagene autoleucel. In some embodiments, the method comprises administering ciltacabtagene autoleucel to a subject, wherein the subject achieves minimum residual disease (MRD) negative status by about 28 days after administration of ciltacabtagene autoleucel.

Description

METHODS OF TREATING SMOLDERING MULTIPLE MYELOMA WITH CILTACABTAGENE AUTOLEUCELCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63 / 712,159, filed on October 25, 2024, U.S. Provisional Application 63 / 729,094, filed on December 6, 2024, and U.S. Provisional Application 63 / 857,162, filed on August 4, 2025, each of which are incorporated herein in their entirety.SEQUENCE LISTING

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “258199.092402 Sequence Listing”, was created on October 24, 2025, and is 24,576 bytes in size.BACKGROUND

[0003] Multiple myeloma is a neoplasm of plasma cells that is aggressive. Multiple myeloma is considered to be a B-cell neoplasm that proliferates uncontrollably in the bone marrow. Symptoms include one or more of hypercalcemia, renal insufficiency, anemia, bony lesions, bacterial infections, hyperviscosity and amyloidosis. Multiple myeloma is still considered to be an almost incurable disease, despite availability of new therapies that include proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies that have significantly improved patient outcomes. Because most patients will either relapse or become refractory to treatment, there is an ongoing need for new therapies for multiple myeloma.

[0004] Smoldering multiple myeloma (SMM) is a precursor condition to multiple myeloma (MM). SMM presents a higher annual risk of progression, particularly within the first five years. High-risk SMM participants may benefit from early therapeutic intervention to prevent irreversible organ damage and symptoms. Early intervention is hypothesized to improve disease response, progression-free survival, and overall survival by preventing clonal evolution and immune-suppressive deregulation. Ongoing research continues to explore novel therapeutic combinations and their roles in high-risk SMM, with the goal of potentially curing or significantly delaying disease progression. Early aggressive treatment targetingprecancerous conditions holds promise for improving outcomes in SMM by delaying or preventing progression to MM.SUMMARY OF THE DISCLOSURE

[0005] Disclosed herein are methods of treating smoldering multiple myeloma in a subject in need thereof. In some embodiments, the method comprises administering ciltacabtagene autoleucel to the subject. In some embodiments, the smoldering multiple myeloma is a high-risk smoldering multiple myeloma.

[0006] In some embodiments, the ciltacabtagene autoleucel is administered to the subject at a dose of about 0.1 to about 1.0 x 106CAR-positive viable T cells / kg. In some embodiments, the ciltacabtagene autoleucel is administered to the subject at a dose of about 0.75 x 106CARpositive viable T cells / kg. In some embodiments, the ciltacabtagene autoleucel is administered to the subject at a dose of about 0.5 x 106CAR-positive viable T cells / kg. In some embodiments, ciltacabtagene autoleucel is administered to the subject at a dose of about 0.3 x 106CAR-positive viable T cells / kg.

[0007] In some embodiments, the ciltacabtagene autoleucel is administered in a single infusion.

[0008] In some embodiments, the maximum total dose of ciltacabtagene autoleucel is 1 x 108CAR-positive viable T-cells.

[0009] In some embodiments, the subject achieves minimum residual disease (MRD) negative status by about 28 days after administration of ciltacabtagene autoleucel. In some embodiments, the subject sustains minimum residual disease (MRD) negative status after about 6 months after administration of ciltacabtagene autoleucel. In some embodiments, the subject sustains minimum residual disease (MRD) negative status after about 12 months after administration of ciltacabtagene autoleucel.

[0010] In some embodiments, the method further comprises treating the subject for an adverse event after administering ciltacabtagene autoleucel, wherein the adverse event comprises a nonhematologic adverse event, a hematologic adverse event, a treatment-emergent adverse event, or any combination thereof. In some embodiments, the adverse event comprises neutropenia, thrombocytopenia, anemia, lymphopenia, hypertriglyceridemia, lymphocytosis, an upper respiratory tract infection, nasopharyngitis, sinusitis, rhinitis, tonsillitis, pharyngitis, laryngitis, pharyngotonsillitis, COVID- 19, COVID- 19 pneumonia, asymptomatic COVID- 19, neutropenic sepsis, progressive multifocal leukoencephalpathy, septic shock, respiratoryfailure, pulmonary embolism, a lower respiratory tract / lung infection, pneumonia, bronchitis, nausea, hypogammaglobulinemia, diarrhea, fatigue, headache, constipation, hypokalemia, asthenia, peripheral edema, decreased appetite, peripheral sensory neuropathy, back pain, arthralgia, pyrexia, dyspnea, insomnia, or any combination thereof. In some embodiments, the nonhematologic adverse event comprises an infection and / or a nonhematologic adverse event other than an infection.

[0011] In some embodiments, the treatment-emergent adverse event comprises cytokine release syndrome (CRS). In some embodiments, the maximum toxicity grade of the CRS is Grade 1 or Grade 2. In some embodiments, the treatment of the CRS comprises intravenous fluids, tocilizumab, methylprednisolone, dexamethasone, oxygen, a corticosteroid, a vasopressor, or any combination thereof.

[0012] In some embodiments, the CAR-T cells in the blood of the subject peak at a median of about 12 days to about 14 days after administering the ciltacabtagene autoleucel to the subject. In some embodiments, the CAR-T cells in the blood of the subject peak at a median of about 14 days after administering the ciltacabtagene autoleucel to the subject. In some embodiments, the CAR-T cells in the blood of the subject peak at a mean concentration of about 6790 cells / pL after administering the ciltacabtagene autoleucel to the subject. In some embodiments, the CAR-T cells in the blood of the subject remain detectable from about 21 days to about 378 days after administering the ciltacabtagene autoleucel to the subject. In some embodiments, the CAR-T cells in the blood of the subject remain detectable at a median of about 70 days after administering the ciltacabtagene autoleucel to the subject. In some embodiments the CAR-T cells in the blood of the subject remain detectable about 3 months after administering the ciltacabtagene autoleucel to the subject. In some embodiments, CAR-T cells in the blood of the subject remain detectable about 6 months after administering the ciltacabtagene autoleucel to the subject. In some embodiments, CAR-T cells in the blood of the subject remain detectable about one year after administering the ciltacabtagene autoleucel to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0014] FIG. 1 shows the design of the ciltacabtagene autoleucel CAR. Ciltacabtagene autoleucel comprises two VHH domains, as opposed to a single VL domain and a single VH domain found on various other CARs. Ciltacabtagene autoleucel comprises intracellular CD 137 and human CD3 zeta domains.

[0015] FIG. 2 shows the expression of BCMA antigen on the surface of GC, memory and plasmablast cells in the lymph node, long-lived plasma cells in the bone marrow LN and MALT, and on multiple myeloma cells. BAFF-R antigen is not expressed on plasmablast cells, long-lived plasma cells, or multiple myeloma cells. TACI is expressed on memory and plasmablast cells, long-lived plasma cells, and multiple myeloma cells. CD138 is expressed only on long-lived plasma cells and multiple myeloma cells.

[0016] FIG. 3 shows a schematic for preparing virus encoding ciltacabtagene autoleucel CAR, transduction of the virus into a T cell from the patient, and then preparation of CAR T cells expressing ciltacabtagene autoleucel.

[0017] FIG. 4 depicts the LCAR-B38M coding sequence (top), comprised of a human CD8 alpha signal peptide (CD8a SP), BCMA targeting domain, human CD8 alpha hinge and transmembrane domain (CD8a hinge+TM), human CD137 cytoplasmic domain, and a human CD3 zeta cytoplasmic domain (CD3Q (FIG. 4). The expression of LCAR-B38M is driven and controlled by a human elongation factor 1 alpha promoter (hEFla promoter). The design of ciltacabtagene autoleucel (bottom), consisting of 2 different VHH (single domain antibody, clone VHH1 and VHH2), features dual targeting domains on BCMA, enabling tight binding of LCAR-B38M to the BCMA-expressing cells.

[0018] An updated Mayo 2018 / IMWG criteria was reported highlighting an additional risk model to predict progression to overt myeloma. This “20-2-20” model uses slightly different thresholds of M-protein (>2gm / dL), sFLC ratio (>20) and / or bone marrow plasmacytosis >20% as independent risk factors. See Table 6. In participants with 2 or more risk factors, the risk of progression at 2 years is 46%, identifying a high-risk population. A subgroup analysis of participants with available cytogenetic data in the Mayo Clinic cohort suggested significant benefit could be achieved by adding tumor genomic markers. The 20-2-2 model also included a risk score tool as shown in FIGs . 5 -6 indicating that a risk of 9- 12 points would indicate about 40% progression at 2 years.

[0019] FIG. 5 depicts a risk score tool based on the Mayo 2018 / IMWG criteria, identifying risk factors for progression to overt myeloma. The table details coefficients, odds ratios, p-values, and assigned scores for various thresholds of FLC Ratio, MC (g / DL), BMPC%, andFISH abnormality. The figure presents a scoring system where higher total risk scores correlate with increased predicted risk of progression at 2 years, with specific scores associated with respective predicted risks and sample percentages.

[0020] FIG. 6 depicts the Mayo 2018 / IMWG risk score tool’s effectiveness in predicting the 2-year progression risk for patients stratified into different risk groups based on their total risk score. The graphs plot the percentage of patients with progression over time, with distinct curves representing low-risk (0-4), low-intermediate risk (5-8), intermediate-risk (9-12), and high-risk (>12) groups. The table summarizes hazard ratios comparing each group to the low -risk reference, highlighting significant increases in progression risk as the total risk score increases.

[0021] FIG. 7 depicts the methodology of a phase II, single-arm study investigating the safety and efficacy of cilta-cel, a BCMA-directed CAR T-cell therapy, in smoldering myeloma patients. The flowchart depicts the process from screening, apheresis, and stem cell collection to lymphodepletion with cyclophosphamide and fludarabine, followed by cilta-cel infusion. Post-infusion monitoring and follow-up are included to assess the primary objectives safety and secondary objectives such as efficacy and progression-free survival.

[0022] FIGs. 8A-8C depict graphs showing CAR+ T cell expansion in patients. FIG. 8A is a plot of total CAR+ T cells as they expand post cilta-cel infusion. FIG. 8B is a plot of CAR+ T cells as a % of total T cells Ruella they expand post cila-cel infusion. FIG. 8C is a plot of CD4+ and CD8+ CAR T cell expansion over 1 year. Collectively, FIGs. 8A, 8B, and 8C show that peak CAR+ cell expansion is at or around 14 days, with CD4+ CAR T cells showing higher levels of expansion compared to CD8+ CAR T cells, although both cell types expand in patients.

[0023] FIG. 9 depicts a plot of the responses of six different patients to cilta-cel therapy for smoldering multiple myeloma. Positive responses to treatment deepen over time, and all patients have achieved a strident complete response (sCR) as of the date of their last follow up.

[0024] FIG. 10 depicts a graph of the levels of kappa light chains in a single patient, both before and after cilta-cel treatment. After cilta-cel infusion, the detectable levels of kappa light chains are near or at zero.DETAILED DESCRIPTION

[0025] The present disclosure provides methods of treating smoldering multiple myeloma in a subject in need thereof. In some embodiments, the method comprises administering ciltacabtagene autoleucel to the subject.

[0026] Several aspects and embodiments of the disclosure are described below, with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the disclosure. One having ordinary skill in the relevant art, however, will readily recognize that the disclosure can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines and animals. The present disclosure is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently 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.

[0027] Any references in the description or in the claims to methods of treatment refer to the compounds, compositions, pharmaceutical compositions and medicaments for use in a method of treatment of the human (or animal) body by therapy (or for diagnosis).

[0028] Any references in the description or in the claims to methods of treatment refer to the use of the compounds, compositions, pharmaceutical compositions for the manufacture of a medicament for the treatment of the human (or animal) body by therapy (or for diagnosis).

[0029] In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.

[0030] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.Definitions

[0031] The terminology used herein is for the purpose of describing particular aspects or embodiments only and is not intended to be limiting. As used herein, the indefinite articles “a”, “an” and “the” should be understood to include plural reference unless the context clearly indicates otherwise.

[0032] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0033] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0034] The term “antibody” includes monoclonal antibodies (including full length 4-chain antibodies or full length heavy-chain only antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab’)2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein. Antibodies contemplated herein include single-domain antibodies, such as heavy chain only antibodies. The terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotype (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen specific TCR), and epitope-binding fragments of any of the above. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U.S. Pat. Appl. Pub. 2007 / 0004909 and Ig -DARTS such as those disclosed in U.S. Pat. Appl. Pub. 2009 / 0060910. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgMl,IgM2, IgAl and IgA2) or subclass. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.

[0035] “Antigen binding fragment” or “antigen binding domain” refers to a portion of an immunoglobulin molecule that binds an antigen. Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include the VH, the VL, the VH and the VL, Fab, F(ab’)2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, VH domains modified to function without a corresponding VL domain, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Patent Publ. Nos. W01998 / 44001, WO 1988 / 01649, WO 1994 / 13804 and WO 1992 / 01047.

[0036] Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway et al. (eds.), Immunobiology, 5thEd., Garland Publishing, New York, N.Y. (2001)). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and 8pher et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246,1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266 Al).

[0037] Phage display can also be used to generate an antibody. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al., supra, and Ausubel etal., supra). Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain. Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Pat. No.6,265,150).

[0038] The antibodies, polypeptides, and proteins of embodiments of the disclosure (including functional portions and functional variants) can be subject to post-translational modifications. They can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt. In some embodiments, they are dimerized or polymerized, or conjugated.

[0039] The antibodies, polypeptides, and / or proteins of embodiments of the disclosure (including functional portions and functional variants thereof) can be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, inc., 2000; and Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Further, some of the antibodies, polypeptides, and proteins of the disclosure (including functional portions and functional variants thereof) can be isolated and / or purified from a source, such as a plant, a bacterium, an insect, a mammal, etc. Methods of isolation and purification are known in the art. Alternatively, the antibodies, polypeptides, and / or proteinsdescribed herein (including functional portions and functional variants thereof) can be commercially synthesized. In this respect, the antibodies, polypeptides, and proteins can be synthetic, recombinant, isolated, and / or purified.

[0040] The terms “B-cell maturation antigen” and “BCMA” as used herein include human B cell maturation antigen, also known as BCMA, CD269, and TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of human BCMA consists, according to UniProt of amino acids 1-54 (or 5-51).

[0041] “Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macacci cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0042] ‘ ‘Cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor.

[0043] “Ciltacabtagene autoleucel” (“cilta-cel”) is a chimeric antigen receptor T cell (CAR-T) therapy comprising two B-cell maturation antigen (BCMA)-targeting VHH domains designed to confer avidity for BCMA. Ciltacabtagene autoleucel can comprise T lymphocytes transduced with the ciltacabtagene autoleucel CAR, a CAR encoded by a lentiviral vector. The CAR targets the human B cell maturation antigen (anti-BCMA CAR). A diagram of the lentiviral vector encoding ciltacabtagene autoleucel CAR is provided in FIG. 1. The amino acid sequence of the ciltacabtagene autoleucel CAR is the amino acid sequence of SEQ ID NO: 17.

[0044] A “chimeric antigen receptor” or “CAR” is an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of at least one antibody (or antibody fragment) linked to T-cell signaling domains. Characteristics of CARs can include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens independent of antigen processing, thus bypassing a major mechanism of tumor evasion. Moreover, when expressed in T-cells, advantageously, CARs do not dimerize with endogenousT cell receptor (TCR) a- and P-chains. T cells expressing a CAR are referred to herein as CAR T cells, CAR-T cells or CAR modified T cells, and these terms are used interchangeably herein. The cell can be genetically modified to stably express at least one antigen-binding domain on its surface, conferring novel antigen specificity that is MHC independent. “BCMA CAR” refers to a CAR having an extracellular binding domain specific for BCMA. “Bi-epitope CAR” refers to a CAR having an extracellular binding domain specific for two different epitopes of an antigen, such as BCMA.

[0045] ‘ ‘Combination” means that two or more therapeutics are administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.

[0046] “Complementarity determining regions” (CDR) are antibody regions that bind an antigen. CDRs may be defined using various delineations such as Kabat (Wu et al. J Exp Med 132: 211-50, 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. J Mol Biol 196: 901-17, 1987), IMGT (Lefranc et al. Dev Comp Immunol 27: 55-77, 2003) and AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996). The correspondence between the various delineations and variable region numbering are described (see e.g., Lefranc et al. Dev Comp Immunol 27: 55-77, 2003; Honegger and Pluckthun, J Mol Biol 309:657-70, 2001; International ImMunoGeneTics (IMGT) database; Web resources, http: / / www_imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The term “CDR”, “HCDR1”, “HCDR2 ”, “HCDR3 ”, “LCDR1”, “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat; Chothia; Martin; Lefranc et al.).Table 1. Kabat, IMGT, AbM, and Chothia numbering systems.IMGT Kabat AbM Chothia VH CDR1 27-38 31-35 26-35 26-32 VHCDR2 56-65 50-65 50-58 53-55 VH CDR3 105-117 95-102 95-102 96-101 VLCDR1 27-38 24-34 24-34 26-32 VLCDR2 56-65 50-56 50-56 50-52VLCDR3 105-117 89-97 89-97 91-96

[0047] “Comprising” is intended to include examples encompassed by the terms “consisting essentially of’ and “consisting of’; similarly, the term “consisting essentially of’ is intendedto include examples encompassed by the term “consisting of.” Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0048] By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. In some embodiments, a “decrease” or “reduced” amount can be a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in-between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage.

[0049] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0050] By ‘ ‘enhance” or “promote,” or “increase” or “expand” or “improve” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in T cell expansion, activation, effector function, persistence, and / or an increase in cancer cell death killing ability, among others apparent from the understanding in the art and the description herein. In some embodiments, an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more times (e.g., 500, 1000 times) (including all integers and decimal points in-between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition. “Enhance” or “enhanced” also refers to enhancement in one or more functions of a test molecule when compared to a control molecule or a combination of testmolecules when compared to one or more control molecules. Exemplary functions that can be measured are tumor cell killing, T cell activation, relative or absolute T cell number, emediated effector function (e.g., ADCC, CDC and / or ADCP) or binding to an Fey receptor (FcyR) or FcRn. “Enhanced” may be an enhancement of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant enhancement.

[0051] The terms “express” and “expression” mean allowing for or causing the information in a gene or DNA sequence to become produced. For example, expression can take the form of producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or transmembrane.

[0052] ‘ ‘Fc gamma receptor” (FcyR) refers to well-known FcyRI, FcyRIIa, FcyRIIb or FcyRIII. Activating FcyR includes FcyRI, FcyRIIa and FcyRIII.

[0053] The terms “fragment of an antibody”, “antibody fragment”, “functional fragment of an antibody”, and “antigen-binding portion” are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1 126-1129 (2005)). The antigen recognition moiety of the CAR encoded by the nucleic acid sequence disclosed herein can contain any BCMA-binding antibody fragment. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol, 16: 778 (1998)) and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between thecomplementary domains on different VH -VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail in, e.g., U.S. Patent Application Publication 2009 / 0093024 Al. Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include portions of an immunoglobulin that bind an antigen, such as the VH, the VL, the VH and the VL, Fab, Fab’, F(ab’)2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, VH domains modified to function without a corresponding VL domain ns, VHH domains, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3, alternative scaffolds that bind an antigen, and multispecific proteins comprising the antigen binding fragments. Antigen binding fragments (such as VH and VL) may be linked together via a synthetic linker to form various types of single antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen binding domain, such as single chain Fv (scFv) or diabody. Antigen binding fragments may also be conjugated to other antibodies, proteins, antigen binding fragments or alternative scaffolds which may be monospecific or multispecific to engineer bispecific and multispecific proteins.

[0054] A “full length antibody” is comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain comprised of subdomains CHI, hinge, CH2 and CH3. Each light chain is comprised of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and the VL may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FW). Each VH and VL is composed of three CDRs and four FW segments, arranged from amino-to-carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3 and FW4.

[0055] The phrase “hazard ratio” refers to a measure of the relative rate of progression to an endpoint as compared to a control group. In outcome-based clinical trials, a reduction in the hazard ratio for a test arm as compared to the control indicates the treatment used in the test arm reduces the risk of the endpoint, in the case of the studies described herein, diseaseprogression or death. Preferably hazard ratio is calculated per a stratified constant piecewise weighted log-rank test.

[0056] The term “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0057] “Human antibody” refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If human antibody contains a constant region or a portion of the constant region, the constant region is also derived from human immunoglobulin sequences. Human antibody comprises heavy and light chain variable regions that are “derived from” sequences of human origin if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci. “Human antibody” typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both. Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases, “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-96, and in Int. Patent Publ. No. W02009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of “human antibody”.

[0058] “Humanized antibody” refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

[0059] ‘ ‘Isolated” refers to a homogenous population of molecules (such as synthetic polynucleotides or a protein such as an antibody) which have been substantially separated and / or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Isolated antibody” refers to an antibody that is substantially free of other cellular material and / or chemicals and encompasses antibodies that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0060] The term “line of therapy,” as used in connection with methods of treatment herein, refers to one or more cycles of a planned treatment program, which may have consisted of one or more planned cycles of single-agent therapy or combination therapy, as well as a sequence of treatments administered in a planned manner. For example, a planned treatment approach of induction therapy followed by autologous stem cell transplantation followed by maintenance is one line of therapy. A new line of therapy is considered to have started when a planned course of therapy has been modified to include other treatment agents or medicaments (alone or in combination) as a result of disease progression, relapse, or toxicity. A new line of therapy is also considered to have started when a planned period of observation off therapy had been interrupted by a need for additional treatment for the disease.

[0061] ‘ ‘Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind one antigenic epitope. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.

[0062] ‘ ‘Mutation” refers to an engineered or naturally occurring alteration in a polypeptide or polynucleotide sequence when compared to a reference sequence. The alteration may be a substitution, insertion or deletion of one or more amino acids or polynucleotides.

[0063] “Newly diagnosed multiple myeloma” (NDMM) refers to the initial identification of multiple myeloma in a patient, which has not been previously diagnosed or treated. Diagnostic criteria for myeloma must be met when the participant was diagnosed. In some embodiments,multiple myeloma is defined as clonal bone marrow plasma cells >10% or biopsy-proven bony or extramedullary plasmacytoma and any one or more of the following myeloma-defining events, as defined by the International Myeloma Working Group (IMWG) diagnostic criteria (“CRAB” criteria):• Evidence of end organ damage that can be attributed to the underlying plasma cell proliferative disorder, specifically:o C: Hypercalcemia: serum calcium >0.25 mmol / L (>1 mg / dL) higher than the ULN or >2.75 mmol / L (>11 mg / dL)o R: Renal insufficiency: CrCl <40 mL / min (measured or estimated by validated equations) or serum creatinine >177 pmol / L (>2 mg / dL) o A: Anemia: hemoglobin value >20 g / L below the lower limit of normal, or hemoglobin value <100 g / Lo B: Bone lesions: >1 osteolytic lesions on skeletal radiography, CT, or PET / CT (If bone marrow has less than 10% clonal plasma cells, more than one bone lesion is required to distinguish from solitary plasmacytoma with minimal marrow involvement; PET / CT=18L-fluorodeoxyglucose PET with CT)• Any one or more of the following biomarkers of malignancy:o Clonal bone marrow plasma cell percentage0>60% (these values are based on the serum Preelite assay [The Binding Site Group, Birmingham, United Kingdom]; the involved FLC must be >100 mg / L)o Involved:uninvolved serum FLC ratio >100 (each focal lesion must be >5 mm in size)o >1 focal lesion on MRI studies (Rajkumar 2014)Clonality should be established by showing K / X-light chain restriction on flow cytometry, immunofluorescence, or IHC. Bone marrow plasma cell percentage should preferably be estimated from a biopsy specimen; in case of a disparity between the aspirate and biopsy, the highest value should be used.

[0064] ‘ ‘Monoclonal Gammopathy of Undetermined Significance” (MGUS) is a plasma cell disorder characterized by the presence of serum M-protein levels less than 3 g / dL, a clonal plasma cell population in the bone marrow constituting less than 10%, and the absence of endorgan damage such as hypercalcemia, renal insufficiency, anemia, and lytic bone lesions, which are collectively referred to as the CRAB criteria. MGUS is considered a precursor condition to more serious plasma cell neoplasms like multiple myeloma (MM) and smolderingmultiple myeloma (SMM). While MGUS itself is typically asymptomatic and does not require immediate treatment, it carries a risk of progression to MM or other related disorders. Studies have shown that individuals with MGUS consistently had the condition in the years preceding the development of MM, with some exhibiting a steady increase in M-protein levels overtime. The prevalence of MGUS increases with age and is higher in men compared to women. Early detection and monitoring of MGUS are crucial for identifying individuals at higher risk of progression to symptomatic myeloma, thereby enabling timely intervention and management.

[0065] “Smoldering Multiple Myeloma” is a plasma cell neoplasm characterized by the presence of serum M-protein levels of 3 g / dE or higher, clonal bone marrow plasma cells constituting 10% or more, and the absence of clinical CRAB criteria — hypercalcemia, renal insufficiency, anemia, and lytic bone lesions. SMM is considered an intermediate stage between monoclonal gammopathy of undetermined significance (MGUS) and symptomatic multiple myeloma (MM). Individuals with SMM have an annual risk of progression to symptomatic MM of approximately 10% for the first five years. High-risk SMM participants, defined by a 50% progression rate within two years, may benefit from early therapeutic interventions to prevent or delay end-organ damage and associated morbidities.

[0066] The term “apheresis” as used herein refers to the art-recognized extracorporeal process by which the blood of a donor or patient is removed from the donor or patient and passed through an apparatus that separates out selected particular constituent(s) and returns the remainder to the circulation of the donor or patient, e.g., by retransfiision. Thus, in the context of “an apheresis material” refers to material obtained using apheresis.

[0067] The phrase “nonresponsive disease” refers to either failure to achieve minimal response or to development of progressive disease while on therapy.

[0068] The terms “nucleic acid”, “nucleotide”, and “polynucleotide” encompass both DNA and RNA unless specified otherwise. By a “nucleic acid sequence” or “nucleotide sequence” is meant the nucleic acid sequence encoding an amino acid; these terms may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by linkers.

[0069] As used herein, the term “operatively linked,” and similar phrases, when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5’ and 3’ UTR, and terminator sequences result in the accurate production of a nucleic acidmolecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0070] ‘ ‘Pharmaceutical composition” refers to composition that comprises an active ingredient and a pharmaceutically acceptable carrier.

[0071] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0072] “Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject.

[0073] “Philadelphia chromosome” or “Ph” refers to a well-known chromosomal translocation between chromosomes 9 and 22, resulting in the oncogenic BCR-ABL gene fusion with constitutively active tyrosine kinase activity. The translocation results in a portion of the BCR gene from chromosome 22qll becoming fused with a portion of the ABL gene from chromosome 9q34, and is designated as t(9;22)(q34;ql 1) under the International System for Human Cytogenetic Nomenclature (ISCN). Depending on the precise location of the fusion, the molecular weight of the resulting fusion protein can range from 185 to 210 kDa. “Philadelphia chromosome” refers to all BCR-ABL fusion proteins formed due the (9;22)(q34;ql 1) translocation.

[0074] The term “protein” or “polypeptide” is used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0075] ‘ ‘Recombinant” refers to DNA, antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means when segments from different sources are joined to produce recombinant DNA, antibodies or proteins.

[0076] ‘ ‘Reduce” or “reduced” refers to a reduction in one or more functions of a test molecule when compared to a control molecule or a combination of test molecules when compared to one or more control molecules. Exemplary functions that can be measured are tumor cell killing, T cell activation, relative or absolute T cell number, Fc-mediated effector function (e.g., ADCC, CDC and / or ADCP) or binding to an Fey receptor (FcyR) or FcRn. “Reduced” may be a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant enhancement.

[0077] The term “refractory,” as used in connection to treatment with a particular treatment agent or medicament or line of therapy herein, refers to diseases or disease subjects that fail to respond to said treatment agent or medicament or line of therapy. “Refractory to a therapy” refers to a cancer that is not amendable to surgical intervention and is initially unresponsive to the therapy. The phrase “refractory myeloma” refers to multiple myeloma that is nonresponsive while on primary or salvage therapy or that has progressed within 60 days of last therapy.

[0078] “Relapsed” refers to a cancer that responded to treatment but then returns.

[0079] The term “single-domain antibody” or “sdAb” refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as “VHHs”. Some VHHs may also be known as “Nanobodies”. A camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0080] As used herein, the terms “specifically binds”, “specifically recognizes”, or “specific for” refer to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as a CAR or a VHH), which is determinative of the presence ofthe target in the presence of a heterogeneous population of molecules including biological molecules.

[0081] The term “specificity” refers to selective recognition of an antigen binding protein (such as a CAR or a VHH) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “multispecific” denotes that an antigen binding protein (such as a CAR or antibody) has two or more antigen-binding sites of which at least two bind different antigen-binding specificities. “Bispecific” as used herein denotes that an antigen binding protein (such as a CAR or antibody) has two different antigen-binding specificities.

[0082] As used herein, the term “subject” refers to an animal. The terms “subject” and “patient” may be used interchangeably herein in reference to a subject. As such, a “subject” includes a human that is being treated for a disease, or prevention of a disease, as a patient. The methods described herein may be used to treat an animal subject belonging to any classification. Examples of such animals include mammals. Mammals, include, but are not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be of the order Carnivora, including felines (cats) and canines (dogs). The mammals may be of the order Artiodactyla, including bovines (cows) and swines (pigs) or of the order Perssodactyla, including equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some embodiments, the mammal is a human.

[0083] The terms “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. As used herein, T cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), a tumor infdtrating cytotoxic T cell (TIL; CD8+ T cell), CD4+CD8+ T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular 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 ap T-cell receptor, but also express a variety of molecular markers that are 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 glycolipid antigens presented by the MHC I -like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Alsoincluded are “gamma-delta T cells (y8 T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated a- and P-TCR chains, the TCR in y8 T cells is made up of a y-chain and a 8-chain. T8 T cells can play a role in immunosurveillance and immunoregulation and were found to be an important source of IL-17 and to induce robust CD8+ cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs”, which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs are typically transcription factor Foxp3 -positive CD4+T cells and can also include transcription factor Foxp3 -negative regulatory T cells that are IL-10-producing CD4+T cells.

[0084] “Therapeutically effective amount” refers to an amount effective, at doses and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic or combination of therapeutics that include, for example, improved well-being of the patient.

[0085] The terms “treat” or “treatment” refer to therapeutic treatment wherein the object is to slow down or lessen an undesired physiological change or disease, or provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., a cessation in the worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and / or remission (whether partial or total and whether detectable or undetectable). “Treatment” can also mean prolonging survival as compared to expected survival if a subject was not receiving treatment. Those in need of treatment include those subjects already with the undesired physiological change or disease as well as those subjects prone to having the physiological change or disease . Treatment may involve a treatment agent, also referred to herein as a “medicament” or “medication,” that may be intended to help achieve the beneficial or desired clinical outcome of interest by its action. Treatment agents or medicaments may be administered to a subject by many routes, including at least intravenous and oral routes. The term “intravenous,” in connection to the administration of treatment agents or medicaments, refers to the administration of said treatment agents or medicaments within one or more veins. The term “oral,” in connection to the administrationof treatment agents or medicaments, refers to the administration of said treatment agents or medicaments via an oral passage such as the mouth.

[0086] ‘ ‘Tumor cell” or a “cancer cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation may arise from infection with a transforming virus and incorporation of new genomic nucleic acid, uptake of exogenous nucleic acid or it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci 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.

[0087] The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain (i.e., variable domain) mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy -chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a P-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the P-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and contribute to the formation of the antigen binding site of antibodies (with the HVRs from the other chain, if the antibody is not a sdAb or HCAb) (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0088] The “variable region” or “variable domain” of an antibody refers to the aminoterminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy -chain only antibodies from the Camelid specieshave a single heavy chain variable region, which is referred to as “VHH” domain. VHH is thus a special type of variable region.

[0089] The dosing frequencies provided for herein are understood to be synonymous with standard terms in the art. For example, “weekly” dosing is understood to be synonymous with “QW”. For example, “biweekly” dosing is understood to be synonymous with “Q2W”. For example, “once every four weeks” is understood to be synonymous with “Q4W”. Unless explicitly stated to the contrary, “once every four weeks” and “monthly” are used interchangeably in the context of dosing frequencies. Accordingly, “monthly” or “once a month” is also understood to be synonymous with “Q4W” unless explicitly stated otherwise.

[0090] When referring to a dosage amount, “pg / kg” or “mg / kg” refers to the amount of an active agent, such as a bispecific antibody or antibody, in microgram (pg) or milligram (mg) administered to a subject per kilogram (kg) body weight of the subject.

[0091] Additionally, throughout this disclosure, various aspects and embodiments of the disclosure can be presented in a range format. 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 disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99 % identity, includes something 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.

[0092] The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated. Antibody constant chain numbering can be found for example at ImMunoGeneTics website, at IMGT Web resources at IMGT Scientific charts.

[0093] The substitutions in the CH3 region are expressed as modified position(s) in the first CH3 domain of the first heavy chain / modified position(s) in the second CH3 domain of the second heavy chain. For example, F405L / K409R refers to a F405L mutation in the first CH3region and K09R mutation in the second CH3 region. L351Y_F405A_Y407V / T394W refers to L351Y, F40FA and Y407V mutations in the first CH3 region and T394W mutation in the second CH3 region. D399FHKRQ / K409AGRH refers to mutation in which D399 may be replaced by F, H, K R or Q, and K409 may be replaced by A, G, R or H.

[0094] Conventional one and three-letter amino acid codes are used herein as shown in Table 2.Table 2. Amino acid abbreviations.Amino acid Three-letter code One-letter codeAlanine Ala AArginine Arg RAsparagine Asn NAspartate Asp DCysteine Cys CGlutamate Gin EGlutamine Glu QGlycine Gly GHistidine His HIsoleucine He ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr TTryptophan Trp WTyrosine Tyr YValine Vai VChimeric Antigen Receptors and Immune Effector Cell Compositions

[0095] International Patent Publication Nos. WO 2018 / 028647 and WO 2022 / 116086 are incorporated by reference herein in their entirety. US Patent No. 10,934,363 is incorporated by reference herein in its entirety.

[0096] The disclosure provides for methods of treating a subject with cells expressing a chimeric antigen receptor (CAR). The CAR comprises an extracellular antigen binding domain comprising one or more single-domain antibodies. In various embodiments, there is provided a CAR targeting BCMA (also referred herein as “BCMA CAR”) comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti -BCMA binding moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain. In someembodiments, the anti-BCMA binding moiety is camelid, chimeric, human, or humanized. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as T cell). In some embodiments, the primary intracellular signaling domain is derived from CD4. In some embodiments, the primary intracellular signaling domain is derived from CD3-zeta. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, 0X40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof. In some embodiments, the co-stimulatory signaling domain is derived from CD 137.

[0097] In some embodiments, the BCMA CAR further comprises a hinge domain (such as a CD8-alpha 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 BCMA CAR further comprises a signal peptide (such as a CD8-alpha signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8-alpha signal peptide, the extracellular antigen-binding domain, a CD8-alpha hinge domain, a CD28 transmembrane domain, a first co-stimulatory signaling domain derived from CD28, a second co-stimulatory signaling domain derived from CD137, and aprimary intracellular signaling domain derived from CD4. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8-alpha signal peptide, the extracellular antigen-binding domain, a CD 8 -alpha hinge domain, a CD 8 -alpha transmembrane domain, a second co-stimulatory signaling domain derived from CD 137, and a primary intracellular signaling domain derived from CD3-zeta. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the BCMA CAR is monovalent.

[0098] The present application also provides CARs that have two or more (including, but not limited to, any one of 2, 3, 4, 5, 6, or more) binding moieties that specifically bind to an antigen, such as BCMA. In some embodiments, one or more of the binding moieties are antigen binding fragments. In some embodiments, one or more of the binding moieties comprise singledomain antibodies. In some embodiments, one or more of the binding moieties comprise a VHH.

[0099] In some embodiments, the CAR is a multivalent (such as bivalent, trivalent, or of higher number of valencies) CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a plurality (such as at least about any one of 2, 3, 4, 5, 6,or more) of binding moieties specifically binding to an antigen (such as a tumor antigen); (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0100] In some embodiments, the binding moieties, such as VHHs (including the plurality of VHHs, or the first VHH and / or the second VHH) are camelid, chimeric, human, or humanized. In some embodiments, the binding moieties or VHHs are connected to each other via peptide bonds or peptide linkers. In some embodiments, each peptide linker is no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long.

[0101] In some embodiments, the first BCMA binding moiety and / or the second BCMA binding moiety is an anti-BCMA VHH. In some embodiments, the first BCMA binding moiety is a first anti-BCMA VHH and the second BCMA binding moiety is a second anti-BCMA VHH.

[0102] In some embodiments, the first anti-BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first anti-BCMA binding moiety comprises a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first anti-BCMA binding moiety comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20.

[0103] In some embodiments, the first anti-BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20.

[0104] In some embodiments, the first BCMA binding moiety comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the first anti-BCMA binding moiety comprises one or more of, or all of, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 2. These sequences correspond to the sequences present in ciltacabtagene autoleucel.

[0105] In some embodiments, the second BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the second BCMA binding moiety comprises a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ IDNO: 22. In some embodiments, the second BCMA binding moiety comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23.

[0106] In some embodiments, the second BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23.

[0107] In some embodiments, the second BCMA binding moiety comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the second BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the second anti-BCMA binding moiety comprises one or more of, or all of, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 4. These sequences correspond to the sequences present in ciltacabtagene autoleucel.

[0108] In some embodiments, the first BCMA binding moiety and the second BCMA binding moiety are connected to each other via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the peptide linker comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 11.

[0109] In some embodiments, the CAR further comprises a hinge domain (such as a CD8-alpha 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 CAR further comprises a signal peptide (such as a CD8-alpha signal peptide) located at the N-terminus of the polypeptide.

[0110] Without wishing to be bound by theory, the CARs that are multivalent, or those CARs comprising an extracellular antigen binding domain comprising a first BCMA binding moiety and a second BCMA binding moiety, may be specially suitable for targeting multimeric antigens via synergistic binding by the different antigen binding sites, or for enhancing binding affinity or avidity to the antigen. Improved avidity may allow for a substantial reduction in the dose of CAR-T cells needed to achieve a therapeutic effect, such as a dose ranging from 4.0 x IO4to 1.0 x 106CAR-T cells per kilogram of the mass of the subject, or 3.0 x 106to 1.0 x 108total CAR-T expressing cells. Monovalent CARs, such as bb2121, may need to be dosed at 5 to 10 times these amounts to achieve a comparable effect. In various embodiments, reduceddosage ranges may provide for substantial reduction in cytokine release syndrome (CRS) and other potentially dangerous side-effects of CAR-T therapy.

[0111] The various binding moieties (e.g., an extracellular antigen binding domain comprising a first BCMA binding moiety and a second BCMA binding moiety) in the CARs described herein may be connected to each other via peptide linkers. The peptide linkers connecting different binding moieties (such as VHHs) may be the same or different. Different domains of the CARs may also be connected to each other via peptide linkers. In some embodiments, the binding moieties (such as VHHs) are directly connected to each other without any peptide linkers.

[0112] The peptide linker in the CARs described herein can be of any suitable length. In some embodiments, the peptide linker is at least about any of 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 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acids to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0113] The CARs of the present application comprise a transmembrane domain that can be directly or indirectly connected to the extracellular antigen binding domain.

[0114] The CAR may comprise a T-cell activation moiety. The T-cell activation moiety can be any suitable moiety derived or obtained from any suitable molecule. In one embodiment, for example, the T-cell activation moiety comprises a transmembrane domain. The transmembrane domain can be any transmembrane domain derived or obtained from any molecule known in the art. For example, the transmembrane domain can be obtained or derived from a CD8a molecule or a CD28 molecule. Without wishing to be bound by theory, CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T- cell receptor (TCR) and is expressed primarily on the surface of cytotoxic T-cells. The most common form of CD8 exists as a dimer composed of a CD8 alpha (CD8a) and CD8 beta (CD80) chain. CD28 is expressed on T-cells and provides co-stimulatory signals required for T-cell activation. CD28 is thereceptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, the CD8a and CD28 are human.

[0115] In addition to the transmembrane domain, the T-cell activation moiety may further comprise an intracellular (i.e., cytoplasmic) T-cell signaling domain. The intercellular T- cell signaling domain can be obtained or derived from a CD28 molecule, a CD3 zeta (Q molecule or modified versions thereof, a human Fc receptor gamma (FcRy) chain, a CD27 molecule, an 0X40 molecule, a 4- IBB molecule, or other intracellular signaling molecules known in the art. Without wishing to be bound by theory: (1) CD28 is a T-cell marker important in T-cell costimulation; (2) CD3 associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs); and (3) 4-1BB, also known as CD137, transmits a potent costimulatory signal to T-cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In a preferred embodiment, the CD28, CD3 zeta, 4- IBB, 0X40, and CD27 are human.

[0116] The T-cell activation domain of the CAR encoded by the nucleic acid sequence disclosed herein can comprise any one of aforementioned transmembrane domains and any one or more of the aforementioned intercellular T-cell signaling domains in any combination. For example, the nucleic acid sequence disclosed herein can encode a CAR comprising a CD28 transmembrane domain and intracellular T-cell signaling domains of CD28 and CD3 zeta. Alternatively, for example, the nucleic acid sequence disclosed herein can encode a CAR comprising a CD8a transmembrane domain and intracellular T-cell signaling domains of CD28, CD3 zeta, the Fc receptor gamma (FcRy) chain, and / or 4-1 BB.

[0117] In some embodiments, the CAR polypeptide further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8-alpha (CD8a SP). In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 9.

[0118] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the transmembrane domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 14.

[0119] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the intracellular signaling domain is derived from CD3 . In some embodiments, the intracellular signaling domain comprises at least one co-stimulatory signaling domains. In someembodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the intracellular signaling domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the intracellular signaling domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 15.

[0120] In some embodiments, the CAR polypeptide further comprises 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 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the hinge domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 13.

[0121] In some embodiments, the CAR comprises a first and a second anti-BCMA binding moiety, wherein the first anti-BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; wherein the second BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; wherein the CAR further comprises: a transmembrane domain derived from CD8a, wherein optionally the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6; a primary intracellular signaling domain derived from CD3 , wherein optionally the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 8; a co-stimulatory signaling domain comprising a cytoplasmic domain of CD 137, wherein optionally the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7; and a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain, wherein the hinge domain is derived from CD8a, wherein optionally the hinge domain comprises the amino acid sequence of SEQ ID NO: 5. In certain such 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.

[0122] In some embodiments, the CAR comprises one or more of, or all of, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23. In some embodiments, the CAR comprises SEQ ID NO: 17. In some embodiments, the CAR comprises a polypeptide encoded by the nucleic acid sequence of one or more of, or all of, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0123] In preferred embodiments, the CAR comprises a first VHH domain comprising a CDR1, a CDR2 and a CDR3 of the VHH domain comprising the amino acid sequence of SEQ ID NO: 2, and a second VHH domain comprising a CDR1, a CDR2 and a CDR3 of the VHH domain comprising the amino acid sequence of SEQ ID NO: 4. In preferred 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. In particularly preferred 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, 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 further preferred embodiments, the CAR comprises a first VHH domain comprising the amino acid sequence of SEQ ID NO: 2, and a second VHH domain comprising the amino acid sequence of SEQ ID NO: 4.

[0124] ‘ ‘Immune effector cells” are immune cells that can perform immune effector functions. In some embodiments, the immune effector cells express at least FcyRIII and perform ADCC effector function. Examples of immune effector cells which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. In some embodiments, the immune effector cells are T cells. In some embodiments, the T cells are autologous T cells. In some embodiments, the T cells are allogeneic 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 upon expressing the CAR and binding to the target cells, such as CD20+ or CD 19+ tumor cells. In some embodiments, the CD8+ T cells lyse antigen-specific target cells upon expressing the CAR and binding to the target cells.

[0125] Biological methods for introducing the vector into an immune effector cell include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.

[0126] Provided herein are dosage forms comprising 3.0 x 107to 1.0 x 108CAR-T cells comprising a CAR comprising a polypeptide provided herein. Provided herein are dosage forms comprising 3.0 x 107to 1.0 x 108CAR-T cells comprising a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first BCMA binding moiety specifically binding to a first epitope of BCMA, and a second BCMA binding moiety specifically binding to a second epitope of BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different. In some embodiments, there are provided dosage forms comprising 3.0 x 107to 1.0 x 108engineered immune effector cells (such as T-cells) comprising a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first anti-BCMA VHH specifically binding to a first epitope of BCMA, and a second anti-BCMA VHH specifically binding to a second epitope of BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different.

[0127] In some embodiments, the dosage form comprises 3.0 x 107to 4.0 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 3.5 x 107to 4.5 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 4.0 x 107to 5.0 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 4.5 x 107to 5.5 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 5.0 x 107to 6.0 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 5.5 x 107to 6.5 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 6.0 x 107to 7.0 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 6.5 x 107to 7.5 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 7.0 x 107to 8.0 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 7.5 x 107to 8.5 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 8.0 x 107to 9.0 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 8.5 x 107to 9.5 x 107of the CAR-T cells. In some embodiments, the dosage form comprises 9.0 x 107to 1.0 x 108of the CAR-T cells.

[0128] In some embodiments, the cell population of the CAR-T dosage forms described herein comprise a T cell or population of T cells, e.g., at various stages of differentiation. Stages of T cell differentiation include naive T cells, stem central memory T cells, central memory Tcells, effector memory T cells, and terminal effector T cells, from least to most differentiated. 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 then 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.

[0129] Naive T cells can have the following expression pattern of cell surface markers:CCR7+, CD62L+, CD45RO-, CD95-. Stem central memory T cells (Tscm) can have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T cells (Tcm) can have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (Tem) can have the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+.Terminal effector T cells (Teff) can 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.

[0130] Further provided by the present application are pharmaceutical compositions comprising any one of the engineered immune effector cells comprising any one of the CARs (such as BCMA CARs) as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing any of the immune effector cells described herein, having the desired degree of purity, with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. In some embodiments, a pharmaceutical composition of CAR-T cells further comprises an excipient selected from dimethylsulfoxide or dextran-40.

[0131] The compositions described herein may be administered as part of a pharmaceutical composition comprising one or more carriers. The choice of carrier will be determined in part by the particular nucleic acid sequence, vector, or host cells expressing the CAR disclosed herein, as well as by the particular method used to administer the nucleic acid sequence, vector, or host cells expressing the CAR disclosed herein. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the disclosure.

[0132] For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives optionally may be used. Thepreservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0133] In addition, buffering agents may 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 optionally may be used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001 % to about 4% by weight of the total composition.

[0134] The composition comprising the nucleic acid sequence encoding the CAR disclosed herein, or host cells expressing the CAR disclosed herein, can be formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) or the nucleic acid sequence disclosed herein to a particular tissue. Liposomes also can be used to increase the half-life of the nucleic acid sequence disclosed herein. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Patents 4,235,871; 4,501,728; 4,837,028; and 5,019,369. The composition can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition disclosed herein occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known to those of ordinary skill in the art. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain composition embodiments of the disclosure.

[0135] In some embodiments, the CAR-T cells are formulated at a dose of about 0.1 x 106cells / kg to about 1.0 x 106cells / kg, of about 0.15 x 106cells / kg to about 0.95 x 106cells / kg, of about 0.2 x 106cells / kg to about 0.90 x 106cells / kg, of about 0.25 x 106cells / kg to about 0.85 x 106cells / kg, and of about 0.3 x 106cells / kg to about 0.80 x 106cells / kg, of about 0.35 x 106cells / kg to about 0.75 x 106cells / kg, and of about 0.4 x 106cells / kg to about 0.70 x 106cells / kg, of about 0.45 x 106cells / kg to about 0.65 x 106cells / kg, and of about 0.5 x 106cells / kg to about 0.60 x 106cells / kg. In some embodiments, the dosage form comprises 0.3 x 106cells / kg. In some embodiments, the dosage form comprises 0.5 x 106cells / kg. In some embodiments, the dosage form comprises 0.75 x 106cells / kg.

[0136] In a preferred embodiment, the dose is formulated at approximately 0.75 x 106cells / kg.

[0137] In a preferred embodiment, the dose is formulated at approximately 0.5 x 106CARpositive viable T-cells / kg.

[0138] In a preferred embodiment, the dose is formulated at approximately 0.3 x 106CARpositive viable T-cells / kg.

[0139] In some embodiments, the CAR-T cells are formulated at a dose of less than 1.0 x 108CAR-T cells per subject.Methods

[0140] The present application further relates to methods and compositions for use in cell immunotherapy. In particular, disclosed herein are methods of treating smoldering multiple myeloma in a subject in need thereof, comprising administering ciltacabtagene autoleucel to the subject.

[0141] In some embodiments, the cell immunotherapy is for treating cancer in a subject, including but not limited to hematological malignancies and solid tumors. In some embodiments, the cell immunotherapy is for treating multiple myeloma in a subject. In some embodiments, the cell immunotherapy is for treating smoldering multiple myeloma in a subject. In some embodiments, the subject is human. In some embodiments, the methods are suitable for treatment of adults and pediatric population, including all subsets of age, and can be used as any line of treatment, including first line or subsequent lines.

[0142] The methods described herein may be used for treating various cancers, including both solid cancer and liquid cancer. In some embodiments, the methods are used to treat multiple myeloma. The methods described herein may be used as a combination therapy with other types of cancer therapies 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, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

[0143] In some embodiments, the cancer is stage I, stage II or stage III, and / or stage A or stage B multiple myeloma based on the Durie-Salmon staging system. In some embodiments, the cancer is stage I, stage II or stage III multiple myeloma based on the International staging system published by the International Myeloma Working Group (IMWG). In some embodiments, the multiple myeloma is progressive.

[0144] In some embodiments, the subject has smoldering multiple myeloma based on the International staging system published by the International Myeloma Working Group(IMWG). In some embodiments, the subject has newly diagnosed multiple myeloma based on the International staging system published by the IMWG. In some embodiments, the subject has standard -risk multiple myeloma based on revised International Staging System (R-ISS) diagnostic criteria. In some embodiments, the subject is stem cell transplant-eligible. In some embodiments, the subject is fit based on IMWG Frailty Index assessment. In some embodiments, the subject is intermediate-fit based on IMWG Frailty Index assessment.

[0145] Any of the anti-BCMA VHHs, CARs, and engineered immune effector cells (such as CAR-T cells) described herein may be used in the method of treating cancer. In some embodiments, the immune effector cells are autologous. In some embodiments, the immune effector cells are allogeneic. In a preferred embodiment, ciltacabtagene autoleucel (“cilta-cel”) CAR-T cells are administered to the subject.

[0146] In some embodiments, apheresis material is collected from the subject for the production of CAR-T cells. In some embodiments, apheresis material is collected from the subject for the production of ciltacabtagene autoleucel.

[0147] In some embodiments, the CAR-T cells are administered at a dose of about 1.0 x 105to 2.0 x 105cells / kg, 1.5 x 105to 2.5 x 105cells / kg, 2.0 x 105to 3.0 x 105cells / kg, 2.5 x 105to 3.5 x 105cells / kg, 3.0 x 105to 4.0 x 105cells / kg, 3.5 x 105to 4.5 x 105cells / kg, 4.0 x 105to 5.0 x 105cells / kg, 4.5 x 105to 5.5 x 105cells / kg, 5.0 x 105to 6.0 x 105cells / kg, 5.5 x 105to 6.5 x 105cells / kg, 6.0 x 105to 7.0 x 105cells / kg, 6.5 x 105to 7.5 x 105cells / kg, 7.0 x 105to 8.0 x 105cells / kg, 7.5 x 105to 8.5 x 105cells / kg, 8.0 x 105to 9.0 x 105cells / kg, 8.5 x 105to 9.5 x 105cells / kg, 9.0 x 105to 1.0 x 106cells / kg, 1.0 x 106to 2.0 x 106cells / kg, 1.5 x 106to 2.5 x 106cells / kg, 2.0 x 106to 3.0 x 106cells / kg, 2.5 x 106to 3.5 x 106cells / kg, 3.0 x 106to 4.0 x 106cells / kg, 3.5 x 106to 4.5 x 106cells / kg, 4.0 x 106to 5.0 x 106cells / kg, 4.5 x 106to 5.5 x 106cells / kg, or 5.0 x 106to 6.0 x 106cells / kg. In some embodiments, the dose comprises approximately 0.3 x 106cells / kg. In some embodiments, the dose comprises approximately 0.5 x 106cells / kg. In some embodiments, the dose comprises approximately 0.75 x 106cells / kg.

[0148] In some embodiments, the CAR-T cells are administered at a dose of less than 1.0 x 108cells per subject. In some embodiments, the CAR-T cells are administered at a dose of about 3.0 to 4.0 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 3.5 to 4.5 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 4.0 to 5.0 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 4.5 to 5.5 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 5.0 to 6.0 x 107cells. In some embodiments, the CAR-T cellsare administered at a dose of about 5.5 to 6.5 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 6.0 to 7.0 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 6.5 to 7.5 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 7.0 to 8.0 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 7.5 to 8.5 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 8.0 to 9.0 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 8.5 to 9.5 x 107cells. In some embodiments, the CAR-T cells are administered at a dose of about 9.0 x 107to 1.0 x 108cells.

[0149] In some embodiments, the dose comprises approximately 0.3 x 106CAR-positive viable T-cells / kg. In some embodiments, the dose comprises approximately 0.5 x 106CARpositive viable T-cells / kg. In some embodiments, the dose comprises approximately 0.75 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered at a dose of about 0.693 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered at a dose of about 0.52 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered at a dose of about 0.94 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered at a dose of about 0.709 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered at a dose of about 0.51 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered at a dose of about 0.95 x 106CAR-positive viable T-cells / kg. In some embodiments, the CAR-T cells are administered in an outpatient setting.

[0150] In some embodiments, the CAR-T cells (e.g., at any of the foregoing doses) are administered in one or more intravenous infusions. In some embodiments, said administration of said CAR-T cells is via a single intravenous infusion. In some embodiments, said single intravenous infusion is administered using a single bag of said CAR-T cells. In some embodiments, said administration of said single bag of said CAR-T cells is completed between the time at which said single bag of CAR-T cells is thawed and three hours after said single bag of CAR-T cells is thawed. In some embodiments, single intravenous administration is administered using two bags of said CAR-T cells. In some embodiments, said administration of each of said two bags of said CAR-T cells is completed between the time at which a first bag of said two bags of CAR-T cells is thawed and three hours after said first bag of CAR-T cells is thawed.

[0151] The composition comprising the host cells expressing the CAR-encoding nucleic acid sequence disclosed herein, or a vector comprising the 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 preferably is 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.

[0152] In some embodiments, prior to the administration of CAR-T cells, subjects may be administered a conditioning regimen.

[0153] Patients undergoing a CAR-T cell therapy may be prepared with a so-called conditioning regimenthat can suppress the patient’s immune system and improve the efficacy of CAR-T cell therapy (Bloo d (2019) 133 (17): 1799-1800).

[0154] The intensity of conventional conditioning regimens can vary significantly. Description of the regimens can refer to genotoxic or non-genotoxic regimens, which may overlap with reference to myeloablative or non-myeloablative regimens. See, for example, Bacigalupo et al. (2009) Biol Blood Marrow Transplant. 15(12): 1628-1633, herein specifically incorporated by reference.

[0155] Myeloablative conditioning regimens are combination of agents expected to produce profound pancytopenia and myeloablation within 1-3 weeks from administration; pancytopenia is long lasting, usually irreversible and in most instances fatal, unless hematopoiesis is restored by hemopoietic stem cell infusion. Examples include total body irradiation and / or administration of high doses of alkylating agents; busulfan, melphalan, cyclophosphamide; etc.

[0156] Non-myeloablative conditioning regiments typically cause minimal cytopenia, and little early toxicity, but are immunosuppressive to the extent that, when followed by administration of an effective dose of HSPC, will result in engraftment of donor lympho-hemopoietic stem cells.

[0157] In certain embodiments the conditioning regimens provided herein are non-myeloablative.

[0158] In some embodiments, the conditioning regimen comprises one or more of cyclophosphamide and / or fludarabine.

[0159] In some embodiments, the conditioning regimen comprises cyclophosphamide administered at a dosage of 300 mg / m2. In some embodiments, the conditioning regimen comprises fludarabine administered at a dosage of 30 mg / m2. In some embodiments, the conditioning regimen comprises cyclophosphamide administered at a dosage of 300 mg / m2and fludarabine at a dosage of 30 mg / m2.

[0160] In some embodiments, the conditioning regimen is administered to the subject daily, for up to 3 days. In some embodiments, the CAR-T therapy is administered to the subject 5 to 7 days after the start of the administration of the conditioning regimen.

[0161] In some embodiments, the collection of apheresis material collected for the production of CAR-T cells, including ciltacabtagene autoleucel, occurs prior to the administration of the conditioning regimen.

[0162] In some embodiments, subjects will undergo apheresis for collection of peripheral blood mononuclear cells (PMBCs). In some embodiments, cilta-cel is generated from T-cells selected from the apheresis.

[0163] In some embodiments, subjects may undergo leukapheresis for approximately 9-14L blood volume to target 9xl09Total White Blood Cells (WBC). In some embodiments, subjects will undergo leukapheresis for approximately 9-14L blood volume to target at least 6xl29Total White Blood Cells (WBC).

[0164] In some embodiments, subjects may undergo a repeat apheresis.

[0165] In some embodiments, subjects will undergo a conditioning regimen of fludarabine and cyclophosphamide prior to receiving cilta-cel treatment. In some embodiments, the conditioning regiment is administered between 7 and 5 days prior to administration of cilta-cel. In some embodiments, the conditioning regimen is continued for up to 3 consecutive days.

[0166] In some embodiments, subjects may be administered a conditioning regimen of IV cyclophosphamide of 300 mg / m2and fludarabine of 30 mg / m2daily.

[0167] In some embodiments, subjects may be administered cyclophosphamide 300 mg / m2via intravenous infusion over 30 minutes followed by fludarabine 30 mg / m2via intravenous infusion over 30 minutes.

[0168] In some embodiments, subjects are treated at a dose of 0.5xl06CAR-positive viable T-cells / kg. In some embodiments, subjects are treated at a dose of 0.75xl06CAR-positive viable T-cells / kg. In some embodiments, subjects are treated at a dose of 0.3xl06CAR-positive viable T-cells / kg.

[0169] In some embodiments, cilta-cel is administered via a single intravenous infusion.

[0170] In some embodiments, subjects receive premedication prior to cilta-cel administration. In some embodiments, subjects receive an antihistamine prior to cilta-cel administration. In some embodiments, subjects receive diphenhydramine prior to cilta-cel administration. In some embodiments, the antihistamine is administered orally approximately 1 hour prior to cilta-cel infusion. In some embodiments, the antihistamine is administered via IV approximately 30 minutes priorto cilta-cel infusion. In some embodiments, subjects receive an antipyretic prior to cilta-cel administration. In some embodiments, subjects receive acetaminophen prior to cilta-cel administration. In some embodiments, the antipyretic is administered orally or via IV approximately 30 minutes priorto cilta-cel administration.

[0171] In some embodiments, subjects could receive supportive therapies. The following are examples of supportive therapies that could be used. Standard supportive care therapies (antiemetics, antidiarrheals, anticholinergics, antispasmodics, antipyretics, antihistamines, analgesics, antibiotics and other antimicrobials, histamine receptor [H2] antagonists or proton pump inhibitors, and other medications intended to treat symptoms or signs of disease) and therapies intended to treat CAR-T cell-related toxicity (i.e., CRS) as clinically indicated, according to institutional standards and as deemed necessary by the investigator. Bisphosphonates could be initiated (if not already being administered) unless contraindicated at least 60 days after cilta-cel infusion and continued until disease progression was established or for at least 2 years. In the case of severe adverse events such as hypercalcemia, bisphosphonates may be administrated as clinically indicated, according to institutional standards and as deemed necessary by the investigator. Transfusion support was permitted to maintain a hemoglobin of >8.0 g / dL (>5mmol / L) as needed, and platelets of >50 x 109 / L until 3 days before the hematology laboratory test, preceding lymphodepletion. Myeloid growth factors were permitted up to 1 day prior to the start of the conditioning regimen. Pegylated myeloid growth factors (i.e., pegfilgrastim) were prohibited. Documented infectious complications were treated with oral or IV antibiotics or other anti-infective agents as considered appropriate by the treating investigator, according to standard institutional practice. Chemotherapy agents used to treat CAR-T cell-related toxicities were permitted upon consultation with the investigator.

[0172] In some embodiments, a subject’s response to the method of treatment is assessed using the International Myeloma Working Group (IMWG)-based response criteria, which are summarized in Table 3. In some embodiments, the response may be classified as a stringent complete response (sCR). In some embodiments, the response may be classified as a completeresponse (CR), which is worse than a stringent complete response (sCR). In some embodiments, the response may be classified as a very good partial response (VGPR), which is worse than a complete response (CR). In some embodiments, the response may be classified as a partial response (PR), which is worse than a very good partial response (VGPR). In some embodiments, the response may be classified as a minimal response (MR), which is worse than a partial response (PR). In some embodiments, the response may be classified as a stable disease (SD), which is worse than a minimal response (MR). In some embodiments, the response may be classified as a progressive disease (PD), which is worse than a stable disease.

[0173] In some embodiments, the method achieves an overall response rate of about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of treated subjects. The overall response rate may be deciphered by calculating the proportion of patients who achieve a partial response, a very good partial response, a complete response, or a stringent complete response.

[0174] In some embodiments, the tests used to assess International Myeloma Working Group (IMWG)-based response criteria are Myeloma protein (M-protein) measurements in serum and urine, serum calcium corrected for albumin, bone marrow examination, skeletal survey and documentation of extramedullary plasmacytomas.

[0175] Non-limiting examples of tests for M-protein measurement in blood and urine are known to one of ordinary skill in the art and comprise serum quantitative Ig, serum protein electrophoresis (SPEP), serum immunofixation electrophoresis, serum FLC assay, 24-hour urine M-protein quantitation by electrophoresis (UPEP), urine immunofixation electrophoresis, and serum p2-microglobulin.

[0176] Calculating serum calcium corrected for albumin in blood samples for detection of hypercalcemia is known to one of ordinary skill in the art. Without wishing to be bound by theory, calcium binds to albumin and only the unbound (free) calcium is biologically active; therefore, the serum calcium level must be adjusted for abnormal albumin levels (“corrected serum calcium”).

[0177] In some embodiments, a skeletal survey of any one of, or all of, the skull, the entire vertebral column, the pelvis, the chest, the humeri, the femora, and any other bones, may be performed and evaluated by either roentgenography (“X-rays”) or low-dose computed tomography (CT) diagnostic quality scans without the use of IV contras, both of which are known to one of ordinary skill in the art. In some embodiments, following T cell administrationand before disease progression is confirmed, X-rays or CT scans may be performed locally, whenever clinically indicated based on symptoms, to document response or progression. In some embodiments, magnetic resonance imaging (MRI) may be used for evaluating bone disease but does not replace a skeletal survey. MRI is known to one of ordinary skill in the art. In some embodiments, if a radionuclide bone scan is used at screening, in addition to the complete skeletal survey, both methods may be used to document disease status. Radionuclide bone scans are known to one of ordinary skill in the art. In some embodiments, the radionuclide bone scan and complete skeletal survey may be performed at the same time. In some embodiments, a radionuclide bone scan may not replace a complete skeletal survey. In some embodiments, if a subject presents with disease progression manifested by symptoms of pain due to bone changes, then disease progression may be documented by skeletal survey or other radiographs, depending on the symptoms that the subject experiences.

[0178] In some embodiments, extramedullary plasmacytomas may be documented by clinical examination or MRI. In some embodiments, if there was no contraindication to the use of IV contrast, extramedullary plasmacytomas may be documented by CT scan. In some embodiments, extramedullary plasmacytomas may be documented by a fusion of positron emission tomography (PET) and CT scans if the CT component is of sufficient diagnostic quality. In some embodiments, assessment of measurable sites of extramedullary disease may be performed, measured, or evaluated locally every 4 weeks for subjects until development of confirmed CR or confirmed disease progression. In some embodiments, evaluation of extramedullary plasmacytomas may be done every 12 weeks.

[0179] In some embodiments, to qualify for VGPR or PR or MR, the sum of products of the perpendicular diameters of the existing extramedullary plasmacytomas may have decreased by over 90% or at least 50%, respectively. In some embodiments, to qualify for disease progression, either the sum of products of the perpendicular diameters of the existing extramedullary plasmacytomas must have increased by at least 50%, or the longest diameter of previous lesion >1 cm in short axis must have increased at least 50%, or a new plasmacytoma must have developed. In some embodiments, to qualify for disease progression when not all existing extramedullary plasmacytomas are reported, the sum of products of the perpendicular diameters of the reported plasmacytomas had increased by at least 50%. In some embodiments, if the study treatment interferes with the immunofixation assay, CR may be defined as the disappearance of the original M-protein associated with multiple myeloma on immunofixation.

[0180] In some embodiments, a subject’s response to the method of treatment is assessed in terms of change in disease burden or tumor burden. Disease burden or tumor burden represents the type of measurable disease in the subject. In some embodiments, the change in tumor burden may be assessed in terms of paraprotein level changes upon treatment. In some embodiments, the paraprotein is an M-protein in the serum. In some embodiments, the paraprotein is an M-protein in the serum. In some embodiments, the change in tumor burden is assessed in terms of the difference between involved and uninvolved free light chain (dFLC). In some embodiments, the change in tumor burden is assessed in terms of the maximum paraprotein reduction from baseline, i.e., from prior to the administration of the CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up time of greater than or equal to 28 days following the administration of CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up time of greater than or equal to 1 month following the administration of CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up time of greater than or equal to 3 months following the administration of CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up time of greater than or equal to 6 months following the administration of CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up time of greater than or equal to 9 months following the administration of CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up time of greater than or equal to 12 months following the administration of CAR-T cells.

[0181] In some embodiments, bone marrow aspirate or biopsy may be performed for clinical assessments or bone marrow aspirate may be performed for biomarker evaluations. In some embodiments, clinical staging (morphology, cytogenetics, and immunohistochemistry or immunofluorescence or flow cytometry) may be done. In some embodiments, a portion of the bone marrow aspirate may be immunophenotyped and monitored for BCMA, checkpoint ligand expression in CD138-positive multiple myeloma cells, and checkpoint expression on T cells. In some embodiments, minimal residual disease (MRD) may be monitored in subjects using next generation sequencing (NGS) of bone marrow aspirate DNA. The NGS of bone marrow aspirate DNA is known to one of ordinary skill in the art. In some embodiments, the NGS is performed via clonoSEQ. In some embodiments, baseline bone marrow aspirates may be used to define the myeloma clones, and post-treatment samples may be used to evaluate MRD negativity. In some embodiments, the MRD negativity status may be based on samplesthat are evaluable. In some embodiments, evaluable samples are those that passed one or more of, or all of, calibration, quality control, and sufficiency of cells evaluable at a particular sensitivity level. In some embodiments, the sensitivity level is 10'6. In some embodiments, the sensitivity level is IO'5. In some embodiments, the sensitivity level is IO'4In some embodiments, the sensitivity level is 10'3.

[0182] In some embodiments, the method achieves MRD-negativity at a threshold of 10'5before disease progression or start of a subsequent antimyeloma therapy. In some embodiments, the method achieves sustained MRD-negative status, as determined by NGS with sensitivity of 10'5, for at least 6 months without examination showing MRD-positive or PD in between.

[0183] Also provided in further embodiments is the medical use of the therapies (including CARs, CAR T cells, and antibodies) provided herein for use in the methods of treatment disclosed herein. Also provided in further embodiments is the use of the therapies (including CARs, and CAR T cells), and antibodies provided herein for use in the manufacture of a medicament for the use in the methods of treatment disclosed herein.Enumerated Embodiments

[0184] The following examples are illustrative, but not limiting, of the compounds, compositions and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.1. A method of treating smoldering multiple myeloma in a subject in need thereof, the method comprising administering ciltacabtagene autoleucel to the subject.2. The method of embodiment 1, wherein the smoldering multiple myeloma is a high-risk smoldering multiple myeloma.3. The method of embodiments 1 or 2, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.1 to about 1.0 x 106CAR-positive viable T cells / kg.4. The method of embodiment 3, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.75 x 106CAR-positive viable T cells / kg.5. The method of embodiment 3, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.5 x 106CAR-positive viable T cells / kg.6. The method of embodiment 3, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.3 x 106CAR-positive viable T cells / kg.7. The method of any one of embodiments 1 to 6, wherein ciltacabtagene autoleucel is administered in a single infusion.8. The method of any one of embodiments 1 to 7, wherein the maximum total dose of ciltacabtagene autoleucel is 1 x 108CAR-positive viable T-cells.9. The method of any one of embodiments 1 to 8, wherein the subject achieves minimum residual disease (MRD) negative status by about 28 days after administration of ciltacabtagene autoleucel.10. The method of embodiment 9, wherein the subject sustains minimum residual disease (MRD) negative status after about 6 months after administration of ciltacabtagene autoleucel.11. The method of embodiment 9, wherein the subject sustains minimum residual disease (MRD) negative status after about 1 year after administration of ciltacabtagene autoleucel.12. The method of any one of embodiments 1 to 8, wherein the method further comprises treating the subject for an adverse event after administering ciltacabtagene autoleucel, wherein the adverse event comprises a nonhematologic adverse event, a hematologic adverse event, a treatment-emergent adverse event, or any combination thereof.13. The method of embodiment 12, wherein the adverse event comprises neutropenia, thrombocytopenia, anemia, lymphopenia, hypertriglyceridemia, lymphocytosis, an upper respiratory tract infection, nasopharyngitis, sinusitis, rhinitis, tonsillitis, pharyngitis, laryngitis, pharyngotonsillitis, COVID-19, COVID-19 pneumonia, asymptomatic COVID-19, neutropenic sepsis, progressive multifocal leukoencephalpathy, septic shock, respiratory failure, pulmonary embolism, a lower respiratory tract / lung infection, pneumonia, bronchitis,nausea, hypogammaglobulinemia, diarrhea, fatigue, headache, constipation, hypokalemia, asthenia, peripheral edema, decreased appetite, peripheral sensory neuropathy, back pain, arthralgia, pyrexia, dyspnea, insomnia, or any combination thereof.14. The method of embodiment 12, wherein the nonhematologic adverse event comprises an infection and / or a nonhematologic adverse event other than an infection.15. The method of embodiment 12, wherein the treatment-emergent adverse event comprises cytokine release syndrome (CRS).16. The method of embodiment 15, wherein the maximum toxicity grade of the CRS is Grade 1 or Grade 2.17. The method of embodiment 15 or 16, wherein treatment of the CRS comprises intravenous fluids, tocilizumab, methylprednisolone, dexamethasone, oxygen, a corticosteroid, a vasopressor, or any combination thereof.18. The method of any one of embodiments 1-17, wherein CAR-T cells in the blood of the subject peak at about 12 days to about 14 days after administering the ciltacabtagene autoleucel to the subject.19. The method of embodiment 18, wherein CAR-T cells in the blood of the subject peak at about 14 days after administering the ciltacabtagene autoleucel to the subject.20. The method of any one of embodiments 1-19, wherein CAR-T cells in the blood of the subject peak at a median absolute CAR+T cell count at peak expansion of about 0.7 K / uL to about 30 K / uL after administering the ciltacabtagene autoleucel to the subject.21. The method of embodiment 20, wherein CAR-T cells in the blood of the subject peak at a median absolute CAR+T cell count at peak expansion of about 3.8 K / uL after administering the ciltacabtagene autoleucel to the subject.22. The method of any one of embodiments 1-21, wherein CAR-T cells in the blood of the subject remain detectable about 3 months after administering the ciltacabtagene autoleucel to the subject.23. The method of any one of embodiments 1-21, wherein CAR-T cells in the blood of the subject remain detectable about 6 months after administering the ciltacabtagene autoleucel to the subject.24. The method of any one of embodiments 1-21, wherein CAR-T cells in the blood of the subject remain detectable about one year after administering the ciltacabtagene autoleucel to the subject.EXAMPLES

[0185] The following examples are provided to further describe some of the aspects and embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed aspects or embodiments.Example 1: B-cell maturation antigen (BCMA) and Ciltacabtagene autoleucel

[0186] B-cell maturation antigen (BCMA, also known as CD269 and TNFRSF17) is a 20 kilodalton, type I membrane protein that is part of the tumor necrosis receptor superfamily. BCMA is a cell surface antigen that is predominantly expressed in B-lineage cells at high levels. BCMA plays a critical role in B cell maturation and subsequent differentiation in plasma cells. BCMA binds 2 ligands: APRIL (CD256) and BAFF receptor (CD257). BCMA is exclusively expressed in mature B-lineage cells and is selectively induced during plasma cell differentiation. FIG. 2 shows the expression of BCMA on various immune -derived cells. Comparative studies have shown a lack of BCMA in most normal tissues and absence of expression on CD34-positive hematopoietic stem cells. BCMA binds 2 ligands that induce B cell proliferation and plays a critical role in B cell maturation and subsequent differentiation into plasma cells. BCMA has been established as a validated target in multiple myeloma with multiple therapeutic modalities demonstrating promising efficacy and acceptable safety profile in heavily pretreated patients. Importantly, BCMA has stable expression on multiple myeloma cells (unlike other markers, such as CD 138) and is a selective cell-surface marker in both multiple myeloma cell lines and samples from patients with multiple myeloma, making it anideal therapeutic target. In addition to expression on the cell surface, BCMA is cleaved by gamma secretase activity at the transmembrane domain, generating serum BCMA (sBCMA) protein. The selective expression and the biological importance for the proliferation and survival of myeloma cells makes BCMA a promising target for CAR-T based immunotherapy.

[0187] Chimeric antigen receptor T (CAR-T) cell therapy uses modified autologous T cells that are activated in an MHC -independent manner upon binding to their target. This results in lysis of the targeted cells. The selective expression and the biological importance for the proliferation and survival of myeloma cells makes BCMA a promising target for CAR-T based immunotherapy.

[0188] Ciltacabtagene autoleucel (also known as cilta-cel) is an autologous chimeric antigen receptor T-cell (CAR-T) therapy that targets BCMA. The ciltacabtagene autoleucel chimeric antigen receptor (CAR) comprises to BCMA-targeting VHH domains designed to confer avidity. Cilta-cel was approved in the US in 2022 based on the CARTITUDE-1 (68284528MMY2001, NCT03548207) study results which demonstrated a 97% ORR in heavily pretreated patients with relapsed / refractory MM. Results in 74 subjects from the Legend-2 study indicate an ORR of 87.8% with a CR rate of 64.9% and results from 97 subjects the CARTTUDE-1 study indicate an ORR of 97.9% with a sCR rate of 80.4%.

[0189] A map of the construct is depicted in FIG. 1 and a schematic of the CAR-T cell production is shown in FIG. 3. Ciltacabtagene autoleucel includes 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. The design features dual targeting domains on BCMA, enabling tight binding of LCAR-B38M to the BCMA-expressing cells. The LCAR-B38M coding sequence is comprised of a human CD8 alpha signal peptide (CD8a SP), BCMA targeting domain consisting of 2 different VHH (single domain antibody, clone VHH1 and VHH2), human CD8 alpha hinge and transmembrane domain (CD8a hinge+TM), human CD137 cytoplasmic domain, and a human CD3 zeta cytoplasmic domain (CD3Q (FIG. 4). The expression of LCAR-B38M is driven and controlled by a human elongation factor 1 alpha promoter (hEFla promoter).Example 2: Multiple Myeloma (MM), Monoclonal Gammopathy of Undetermined Significance (MGUS), and Smoldering Multiple Myeloma (SMM)

[0190] Multiple myeloma (MM) is a plasma cell neoplasm characterized by multifocal proliferation of clonal, long-lived plasma cells associated with an overproduction ofmonoclonal gamma globulin. The International Myeloma Working Group (IMWG) has defined monoclonal gammopathy of undetermined significance (MGUS) as the presence of serum M-protein < 3g / dL, clonal plasma cell population in the bone marrow < 10%, and the absence of end-organ damage such as hypercalcemia (serum calcium > 11.5 mg / dL), renal insufficiency (serum creatinine > 2 mg / dL), anemia (hemoglobin value below the lower limit of normal by more than 2 g / dL or hemoglobin value < 10 g / dL), and lytic bone lesions (CRAB criteria) that can be attributed to the plasma cell proliferative disorder. Smoldering multiple myeloma (SMM) was defined by serum M-protein > 3 g / dL, clonal bone marrow plasma cells > 10%, and the absence of clinical CRAB criteria. See Table 4.

[0191] It has been known that some cases of MGUS progressed to symptomatic myeloma, but it was not clear whether all cases of myeloma were preceded by MGUS. In a study of more than 77,000 individuals, 55 to 74 years of age, from a cancer screening trial, Landgren et al. (2013) found 71 participants who developed MM. The study showed that participants who eventually developed MM consistently had MGUS in the years preceding development of MM. Interestingly, in half the participants, there was a steady increase in the M-protein before the development of symptomatic myeloma, while in the other half the M-protein stayed fairly stable. A second study by Weiss et al. (2009) found a monoclonal gammopathy in 27 of 30 participants (90%) in sera 2 or more years before the diagnosis of myeloma.

[0192] The diagnosis of smoldering (asymptomatic) multiple myeloma (SMM) is based on the demonstration of M-protein in serum (>3 gm / dL) or urine and / or the presence of 10-60% clonal bone marrow plasma cells (BMPC). Participants with SMM have an annual risk of progression of 10% for the first 5 years. As such, SMM is thought to comprise a mixture of high-risk participants, who may benefit from early therapeutic intervention before irreversible organ damage and symptoms appear. These high-risk SMM participants are defined as having a 50% progression rate within 2 years. It is hypothesized that early therapeutic interception of disease progression in these high-risk individuals will prevent / delay end-organ damage with all its morbidities. In addition, early intervention before clonal evolution with secondary acquisition of mutations along with immune-suppressive deregulation of the microenvironment would likely lead to a significant improvement in disease response, progression-free survival, and overall survival.

[0193] In a large population-based study, Kyle et al. (2006) analyzed serum samples of more than 75% of residents, 50 years or older. They identified MGUS in 694 of 21,463 participants tested (3.2%). While the overall prevalence was noted to be 3.2% (95 % CI, 3.0 to 3.5), therewas a significant age dependent increase in both sexes with the prevalence among persons 80 years of age or older 4 times as high as among those 50-59 years of age. Age-adjusted rates were higher in men (4.0 %, 95% C.I., 3.5 to 4.4) than in women (2.7%, 95% C.I., 2.4 to 3.0). In a subsequent study on a majority of the same participants from Kyle et al., Dispenzieri, et al. (2010) used the free light chain assay (FREELITE) and showed that 0.8% of people older than 50 years had light chain-MGUS. The total MGUS prevalence including the light chain-MGUS cases was noted at 4.2% (95% C.I., 3.9 to 4.5%). A limitation of both these studies was that 97.3% of the residents of Olmsted County were white.

[0194] For SMM participants, a M-protein > 3 g / dL, a FLC ratio outside the range of 0.125 to 8, and > 10% plasma cells in the bone marrow are considered as adverse factors in this model. See Table 5. The 5 -year rate of progression in participants with 1, 2 and 3 risk factors was 25%, 51% and 76% respectively. The time to progression with these risk factors was 10, 5.1, and 1.9 years, respectively. Rajkumar et al. (2011) have proposed that SMM with >60% plasma cells progress to multiple myeloma within 2 years in 95% cases and should be treated at diagnosis even in the absence of symptoms.

[0195] An updated Mayo 2018 / IMWG criteria was reported highlighting an additional risk model to predict progression to overt myeloma. This “20-2-20” model uses slightly different thresholds of M-protein (>2gm / dL), sFLC ratio (>20) and / or bone marrow plasmacytosis >20% as independent risk factors. See Table 6. In participants with 2 or more risk factors, the risk of progression at 2 years is 46%, identifying a high-risk population. A subgroup analysis of participants with available cytogenetic data in the Mayo Clinic cohort suggested significant benefit could be achieved by adding tumor genomic markers. The 20-2-2 model also included a risk score tool as shown in FIGs . 5 -6 indicating that a risk of 9- 12 points would indicate about 40% progression at 2 years.

[0196] Recent studies have significantly advanced understanding of the biology and treatment of smoldering multiple myeloma (SMM) and its progression to multiple myeloma (MM). A study by Kyle et al. (2019) demonstrated that genomic markers such as MAPK pathway mutations and Myc alterations can enhance predictive modeling for SMM progression. By employing whole-exome sequencing and targeted sequencing on 214 SMM samples, the study found that 80% showed somatic copy number alterations, and significant proportions exhibited driver chromosomal translocations, single nucleotide variations, and pathway mutations, with NRAS, KRAS, and BRAF mutations being the most prevalent. Thedata indicated shorter median time to progression in participants harboring MY C oncogene aberrations and MAPK pathway mutations.

[0197] Single-cell RNA sequencing (scRNA-seq) and mass cytometry studies by Bailur et al. (2019) and Zavidii et al. (2020) revealed immune dysregulation in the bone marrow tumor microenvironment of SMM and MGUS (monoclonal gammopathy of undetermined significance) participants, including alterations in memory cytotoxic T cells and the presence of regulatory T cells. These findings highlight the potential for targeted immunotherapeutic interventions in early MM stages.

[0198] Das et al. (2016) found that MM disease progression could be facilitated by tumor-extrinsic factors, as shown by rapid disease progression in a humanized mouse model upon transplantation of tumor cells from latent disease participants. T cell immunity against sternness-related antigens and regulatory T cell increases were also shown to be significant predictors of disease progression in studies by Dhodapkar et al. (2015) and Kawano et al. (2018).

[0199] Several studies have confirmed that early immunotherapy can play a critical role in altering the natural history of SMM. Mateos et al. (2013) demonstrated that early intervention with lenalidomide and dexamethasone significantly increased progression-free survival (PFS) and overall survival in high-risk SMM patients. Similarly, Lonial et al. (2020) showed that lenalidomide significantly improves PFS across various risk groups. Additional trials focusing on more potent therapies, such as the GEM-CESAR trial using a combination of carfilzomib, lenalidomide, and dexamethasone with stem cell transplantation, have shown promising results in high-risk SMM participants by achieving high rates of minimal residual disease negativity and prolonged progression-free periods.

[0200] Ongoing research continues to explore novel combinations and their roles in high-risk SMM with a view toward potentially curing or significantly delaying disease progression. The effectiveness of immunotherapy in preventing MM progression is also investigated through studies on CAR-T cells and bispecific antibodies, showing promising responses.

[0201] In sum, early aggressive treatment targeting precancerous conditions holds promise for improving outcomes in SMM by delaying or even preventing progression to MM, highlighting the importance of further exploration and validation of these therapeutic strategies. The goal of therapy in high-risk SMM should be to deliver as highly effective therapy as possible to ultimately achieve cure or significant delay in PFS without compromising safety. The use of CAR-T cell therapy in this high-risk population would not constitute a moreaggressive approach and would not be any more toxic compared to currently used interventions.Example 3: A Chimeric Antigen Receptor T Cell (CAR-T) Therapy Directed Against BCMA in High-Risk Smoldering Myeloma

[0202] Study Rationale

[0203] The rationale for the present study is that early therapeutic interception of disease progression in the high-risk individuals described in Example 2 could prevent / delay end-organ damage with all its morbidities. In addition, early intervention before clonal evolution with secondary acquisition of mutations along with immune-suppressive deregulation of the microenvironment was hypothesized to lead to a significant improvement in disease response, progression-free survival, and overall survival.

[0204] Study Design

[0205] The goal of therapy in high-risk Smoldering Multiple Myeloma (SMM) is to deliver as highly effective therapy as possible to ultimately achieve cure or significant delay in PFS without compromising safety. The use of CAR-T cell therapy in this high-risk population would not constitute a more aggressive approach and would not be any more toxic compared to currently used interventions.

[0206] This study had a first safety run-in cohort of three participants in a staggered fashion, whereby the preceding patient was at least 6 weeks apart before the subsequent patient could be treated. A safety review committee reviewed safety data after the first 3 subjects had the opportunity to complete the dose-limiting toxicity (DLT) window of 60 days. Following safety review of the first cohort, the second cohort were treated with similar criteria. Once the safety run-in was completed, an additional expansion cohort of 14 participants will be treated.

[0207] During the screening phase, all subjects provided written consent for study participation and were screened for study eligibility within 28 days prior to determining eligibility for the study and clearance for apheresis.

[0208] Eligible subjects underwent apheresis for collection of peripheral blood mononuclear cells (PBMC). Study enrollment was defined as the day of apheresis. Cilta-cel was generated from T cells selected from the apheresis. Subjects for whom apheresis or manufacturing failed were allowed a second attempt at apheresis.

[0209] After meeting safety criteria for treatment, subj ects were administered a conditioning regimen of IV cyclophosphamide 300 mg / m2and fludarabine 30 mg / m2daily for 3 days. Cilta-cel was administered at a total targeted dose as described below 5 to 7 days after start of the conditioning regimen.

[0210] For the primary efficacy analysis, disease status was evaluated according to clinical judgement guided by the IMWG consensus recommendations for multiple myeloma treatment response criteria. Safety evaluations included a review of adverse events, laboratory test results, vital sign measurements, physical examination findings, handwriting assessments, and assessment of Eastern Cooperative Oncology Group (ECOG) performance status grade. The safety profile was evaluated in the first 6 participants. Follow-up of subjects for disease progression continued during the post-treatment phase. All study evaluations were conducted according to the Schedule of Events, as presented in Tables 7-9. See Table 7 for all screening requirements and testing that may occur outside of the 28-day screening window. See Table 9 for collection of all PK / biomarker sampling timepoints. See Table 8 for all follow-up procedures. The first analysis was conducted approximately 6 months after the last subject received their initial dose of cilta-cel. Following completion of the study, the investigator will monitor participants treated with cilta-cel for up to 15 years after last administration of cilta-cel for safety outcomes including second primary malignancies, survival status, and disease status according to standard of care.

[0211] Study Objectives

[0212] The primary objective of this study was to determine the safety of CAR-T therapy in high-risk smoldering multiple myeloma (SMM) participants.

[0213] The secondary objectives of this study included the following: to assess proportion of high-risk smoldering multiple myeloma participants who achieve complete remission (CR) or better after beginning treatment with the investigational agents; to assess rate of minimal residual disease (MRD) negativity at 6 months, 1 year, and 2 years; to assess progression-free survival until progression to myeloma; to assess the progression-free survival post initiation of active myeloma therapy (PFS2); to assess time to progression; to assess duration of response; to evaluate and explore pharmacodynamic biomarkers of antimyeloma and immune activity of cilta-cel; and to assess the immunogenicity of cilta-cel in SMM.

[0214] Participant Selection

[0215] All consented participants entered the screening phase for up to 28 days from consent date and eligibility was determined within this window. Once determined eligible, participants were cleared for apheresis to be completed within 40 days from the date of eligibility determination, except as noted in the relevant Schedule of Events. See Tables 7-9.

[0216] Both men and women of all races and ethnic groups were eligible for this trial.

[0217] All participants agreed not to donate blood during therapy, were willing to be hospitalized or remain in close proximity (within 2 hours) to the hospital for 28 days after administration of the CAR-T therapy as described in Table 10 and were willing to be hospitalized following specified AEs as described in Table 10.

[0218] The inclusion criteria for the study were as follows.

[0219] Participants must have been over 18 years of age. They must have had high-risk smoldering multiple myeloma (SMM) with 40% or fewer plasma cells in the bone marrow and have met one of the following high-risk criteria. They must have either meet the "20-2-20" criteria, which was defined by the presence of any two of the following: serum M-protein levels of 2 gm / dL or higher, an involved to uninvolved free light chain (FLC) ratio of 20 or higher, or bone marrow plasma cells (BMPC) percentage between 20% and less than 40%. Alternatively, they could have had a total score of 9 using the following scoring system: FLC ratio (10-25 = 2 points, 25-40 = 3 points, greater than 40 = 5 points), serum M-protein (1.5-3 g / dL = 3 points, greater than 3 g / dL = 4 points), BMPC percentage (15-20% = 2 points, 20-30% = 3 points, 30-40% = 5 points, greater than 40% = 6 points), and FISH abnormality (t(4,14), t(14,16), Iq gain, or dell 3q = 2 points).

[0220] They must have had 10% or more BMPC and at least one of the following: an evolving pattern (e.g., a 10% or greater increase in serum M-protein over a 6-month period, an evolving change in hemoglobin of 0.5 g / dL or greater decrease over a 12-month period, or a progressive involved light chain increase of more than 10% over a 6-month period with a light chain ratio greater than 8), an abnormal PC immunophenotype (95% or more of BMPCs are clonal) with a reduction of one or more uninvolved immunoglobulin isotypes (only IgG, IgA, and IgM will be considered), high-risk cytogenetics (presence of t(4;14), t(14; 16), t(14;20), 17p deletion, TP53 mutation, lq21 gain, or 13q deletion), or monoclonal light chain excretion of more than 200mg / 24 hours for those with monoclonal light chain smoldering multiple myeloma. Patients with biclonal smoldering myeloma were included and followed by their dominant clone for response.

[0221] Participants must have not shown evidence of CRAB criteria or new criteria of active multiple myeloma (SLIM-CRAB), which includes increased calcium levels (corrected serum calcium greater than 0.25 mmol / L or Img / dL above the upper limit of normal or greater than 2.75 mmol / L or llmg / dL), renal insufficiency attributable to myeloma, anemia (hemoglobin 2g / dL below the lower limit of normal or less than lOg / dL), bone lesions (lytic lesions orgeneralized osteoporosis with compression fractures), bone marrow plasma cells greater than 60%, serum involved / uninvolved FLC ratio of 100 or higher (note: in light chain myeloma, a serum involved / uninvolved FLC ratio greater than 100 was not considered a myeloma-defining event if urinary monoclonal protein is less than 200mg / 24 hours), or PET / CT and / or MRI with more than one focal lesion. Participants with CRAB criteria attributable to conditions other than the disease under study were eligible after discussion with the Sponsor Investigator.

[0222] Participants must have had an ECOG Performance Status of 0 or 1.

[0223] Laboratory values obtained within 28 days prior to registration must have included an absolute neutrophil count (ANC) greater than 1000 / mL, platelet count greater than 75,000 / mL, total bilirubin less than or equal to 2.0 mg / dL (if total is elevated, check direct and if normal, the patient is eligible), AST less than or equal to 2.5 times the institutional upper limit of normal (ULN), ALT less than or equal to 2.5 times the institutional ULN, and estimated creatinine clearance (CrCl) of 60 mL / min or higher (Cockcroft Gault equation) or serum creatinine less than or equal to 1.5 times ULN.

[0224] Participants must have provided voluntary written informed consent before any study-related procedures not part of normal medical care, with the understanding that consent may be withdrawn at any time without prejudice to future medical care.

[0225] Women of childbearing potential must have had a negative pregnancy test at screening and agreed to practice a highly effective method of contraception (failure rate of less than 1% per year when used consistently and correctly) from the time of signing the informed consent form until one year after receiving a cilta-cel infusion. Sexually active males agreed to use a barrier method of contraception (e.g., condom with spermicidal foam / gel / film / cream / suppository) from the time of signing the informed consent form until one year after receiving a cilta-cel infusion if sexually active with a woman of childbearing potential. Both women and men agreed not to donate eggs (ova, oocytes) or sperm, respectively, during the study and for one year after the last dose of study treatment.

[0226] The exclusion criteria were as follows.

[0227] Participants must not have received prior SMM-directed therapy within six months of beginning treatment on the study. They should not have had symptomatic multiple myeloma or any evidence of CRAB criteria, including the presence of myeloma-defining events (MDE). Any prior therapy for active myeloma was also excluded, although bisphosphonates were allowed. Participants must not have been undergoing other concurrent chemotherapy, immunotherapy, radiotherapy, or any ancillary therapy considered investigational. Priortherapy with bisphosphonates was allowed, as was prior radiation therapy to a solitary plasmacytoma, provided it was administered at least one year prior to enrollment in the trial.

[0228] Participants with serious medical or psychiatric illnesses likely to interfere with participation in the clinical study were excluded. Those diagnosed or treated for another malignancy within two years of enrollment were also excluded, with exceptions for complete resection of basal cell carcinoma or squamous cell carcinoma of the skin, an in-situ malignancy, or low-risk prostate cancer after curative therapy. Uncontrolled intercurrent illnesses, including but not limited to ongoing or active infection, symptomatic congestive heart failure, autoimmune disease, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness / social situations that would limit compliance with study requirements, were grounds for exclusion.

[0229] Participants who planned to father a child while enrolled in the study or within one year after receiving the last dose of the study drug were excluded. Pregnant or breastfeeding women, or those planning to become pregnant while enrolled in the study or within one year after receiving the last dose of the study drug, were also excluded. Known seropositive individuals orthose with active viral infections such as HIV, hepatitis B, hepatitis C, or SARS-CoV-2 (COVID- 19) were excluded, with specific conditions: participants seropositive due to hepatitis B vaccine are eligible, those positive for HIV1 and 2 antibody, hepatitis B core antibody, or hepatitis B surface antigen must have had a negative PCR result before enrollment, and those positive for SARS-CoV-2 antibody must have had a negative rapid antigen or PCR result. Participants with undetectable viral load and on stable antiretrovirals for HIV1 or 2 infections, or those with past HCV infection that has cleared, were not excluded.

[0230] Participants with contraindications or life-threatening allergies, hypersensitivity, or intolerance to any study drug or its excipients were excluded. Prior or concurrent exposure to specific therapies, including Teclistamab, Belantamab, any anti-BCMA therapy, investigational vaccines within four weeks of registration, live attenuated vaccines within four weeks of registration, monoclonal antibody therapy within 21 days (except for those unrelated to MM therapy such as rituximab or other monoclonal antibodies for RA), cytotoxic therapy within 14 days of registration, PI therapy within 14 days of registration, IMiD agent therapy within 14 days of registration, and radiotherapy within 14 days or focal radiation within seven days of registration, were grounds for exclusion.

[0231] Participants with known active CNS involvement or clinical signs of meningeal involvement of multiple myeloma were excluded. If either was suspected, a negative whole brain MRI and lumbar cytology were required. Those with myelodysplastic syndrome or activemalignancies (i.e., progressing or requiring treatment change in the last 24 months) were excluded, with exceptions for non-muscle invasive bladder cancer treated within the last 24 months and considered completely cured, skin cancer (non-melanoma or melanoma) treated within the last 24 months and considered completely cured, noninvasive cervical cancer treated within the last 24 months and considered completely cured, localized prostate cancer (NOMO) with a Gleason score of less than 6 treated within the last 24 months or untreated and under surveillance, localized prostate cancer with a Gleason score of 3 or 4 treated more than six months prior to study screening and considered to have a very low risk of recurrence, history of localized prostate cancer receiving androgen deprivation therapy and considered to have a very low risk of recurrence, adequately treated lobular carcinoma in situ or ductal carcinoma in situ, history of localized breast cancer receiving anti-hormonal agents and considered to have a very low risk of recurrence, or other malignancy considered cured with minimal risk of recurrence in the judgment of the investigator.

[0232] Participants with a history of stroke or seizure within six months prior to signing the informed consent form were excluded. Those with specific cardiac conditions, including New York Heart Association stage III or IV congestive heart failure, myocardial infarction or coronary artery bypass graft within six months, history of clinically significant ventricular arrhythmia or unexplained syncope not believed to be vasovagal in nature or due to dehydration, or history of severe non-ischemic cardiomyopathy, were also excluded.

[0233] Participants who had undergone major surgery within two weeks prior to registration, had not fully recovered from surgery, or had major surgery planned during the study period or within two weeks after administration of the last dose of study treatment were excluded. Those with concurrent medical or psychiatric conditions or diseases likely to interfere with study procedures or results, or that in the opinion of the investigator would constitute a hazard for participating in the study, were also excluded. This included uncontrolled diabetes, acute diffuse infiltrative pulmonary disease, evidence of active systemic viral, fungal, or bacterial infection requiring systemic antimicrobial therapy, history of autoimmune disease (with exceptions for vitiligo, type I diabetes, and prior autoimmune thyroiditis currently euthyroid based on clinical symptoms and laboratory testing), disabling psychiatric conditions (e.g., alcohol or drug abuse), severe dementia, altered mental status, or any other issue impairing the ability to receive or tolerate the planned treatment, understand informed consent, or any condition for which participation would not be in the best interest of the participant or could prevent, limit, or confound the protocol-specified assessments.Participants with a history of non-compliance with recommended medical treatments were also excluded.

[0234] Managing physicians may allow, at their discretion and approval from the sponsorinvestigator, patients to be included in this study that do not otherwise strictly meet the inclusion criteria disclosed herein. Non-limiting examples of possible inclusion criteria discretion follow:• Certain tests, such as bone marrow biopsies, PET / CT scans, and other screening assessments may be performed on a patient as part of an ongoing standard of care regime. Such tests and / or their results that are performed or obtained prior to the patient’s written consent for this study may not need to be repeated within the screening window at the discretion of the managing physician if the tests were deemed recent and their results were in compliance with the inclusion criteria.• Certain infectious diseases tests, e.g., Hepatitis B surface antibody, Hepatitis B surface antigen, Hepatitis B core antibody, Hepatitis C antibody, and / or HIV antibody, may be drawn prior to apheresis even though the results may not be available until after apheresis. The patient may remain in the study as long as all infectious disease tests are negative.• As described herein, the creatinine clearance (CrCl) threshold for inclusion is 60 mL / min or higher, while the CrCl exclusion via CRAB criteria is lower than 40 mL / min. A patient with a CrCl result slightly lower than 60 mL / min, but higher than 40 mL / min may be included in the study at the discretion of the managing physician if the lower CrCl score is likely due to a low body weight and not renal failure.

[0235] Pharmaceutical Information

[0236] The chemical name for fludarabine phosphate is 9H-Purin-6-amine, 2-fluoro-9-(5-0- phosphono- -D-arabino-furanosyl) (2-fluoro-ara-AMP). The molecular formula of fludarabine phosphate is CI0H13FN5O7P. Its molecular weight is 365.2. Lludarabine is supplied as a white, lyophilized solid cake. Each vial contains 50 mg of fludarabine phosphate, 50 mg of mannitol, and sodium hydroxide to adjust pH to 7.7. The pH range for the final product is 7.2-8.2. Lludarabine is supplied in a clear glass single dose vial (6mL capacity) and packaged in a single dose vial carton in a shelf pack of five. Fludarabine phosphate is stored under refrigeration, between 2°-8°C (36°-46°F).

[0237] Cyclophosphamide is a white crystalline powder with the molecular formula C7H15C12N2P H2O and a molecular weight of 279.1. The chemical name forcyclophosphamide is 2-[bis(2 -chloroethyl) amino tetrahydro-2H-l,3,2- oxazaphosphorine 2-oxide monohydrate. Cyclophosphamide is commercially available for parenteral injection as 100 mg, 200 mg, 500 mg, 1 g, and 2 g vials. Unopened vials of cyclophosphamide are stable until the date indicated on the package when stored at or below 25°C (77°F).

[0238] Cilta-cel is a BCMA-directed genetically modified autologous T-cell immunotherapy. Cilta-cel is prepared from the patient’s peripheral blood mononuclear cells, which are obtained via standard apheresis procedures. The mononuclear cells are enriched for T-cells and genetically modified ex vivo by transduction with a replication-incompetent lentiviral vector to express a chimeric antigen receptor (CAR) comprising an anti-BCMA targeting domain, which consists of two single-domain antibodies linked to a 4- IBB costimulatory domain and a CD3-zeta signaling domain.

[0239] The transduced anti-BCMA CAR-T cells are expanded in cell culture, washed, formulated into a suspension and cryopreserved. The product must pass a sterility test before release for shipping as a frozen suspension in a patient-specific infusion bag. The product is thawed and then infused back into the patient, where the anti-BCMA CAR-T cells can recognize and eliminate BCMA -expressing target cells.

[0240] Initial apheresis material to be used in ciltacabtagene autoleucel manufacturing targeted an autologous mononuclear cell (MNC-A) collection of9xl09WBC (acceptable range 6'12X109WBC) with possibility of 2ndcollection of apheresis collection or subsequent manufacturing if final product was not sufficient for release.

[0241] Ciltacabtagene autoleucel product was shipped for storage in liquid nitrogen under monitored conditions. Once the patient was deemed eligible for infusion, the ciltacabtagene autoleucel was thawed for dosing at an overall study target of 0.75xl06CAR T cells / kg (range in product bag may be 0.5-1.0 xlO6CAR T cells / kg, maximum total dose IxlO8CAR T cells). To achieve doses required for Cohort One / Dose Level -1 or Dose Level -2, the product volume was calculated to meet required dose based on data provided on ciltacabtagene autoleucel product certificate of analysis (COA) under aseptic processing conditions in an ISO-5 Biosafety Cabinet.

[0242] Ciltacabtagene autoleucel was stored in vapor phase of liquid nitrogen (< -120°C) storage prior to thawing (at 37°C + 2 degrees) and infusion (at 20-25°C). Initial apheresis material could be used for subsequent manufacturing until 6 months from collection and ciltacabtagene autoleucel could be infused for up to 9 months after manufacturing.

[0243] Cilta-cel was provided in a in 30 mL or 70mL infusion bags to comprise the correct cell dose containing CAR-positive viable T-cells based on the subject weight reported at the time of apheresis.

[0244] The product was thawed in a water bath (at 37°C + 2 degrees) and infused IV at 20-25°C by gravity or IV pump without any in-line leukocyte-reducing fdter, followed by a normal saline flush. If multiple investigational product (IP) bags were to be administered, only one IP administration was prepared at a time.

[0245] Cytokine Release Syndrome (CRS), including fatal or life-threatening reactions, can occur in participants following treatment with cilta-cel. Severe or life-threatening CRS was treated per Table 3. Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS), which may be fatal or life-threatening, can occur following treatment with cilta-cel, including before CRS onset, concurrently with CRS, after CRS resolution, or in the absence of CRS. Neurologic events after treatment with cilta-cel were monitored and supportive care was provided per Table 11. Parkinsonism and Guillan-Barre Syndrome and their associated complications resulting in fatal or life-threatening reactions have occurred following treatment with cilta-cel. Hemophagocytic Lymphohistiocytosis / Macrophage Activation Syndrome (HLH / MAS) including fatal and life-threatening reactions have occurred in participants following treatment with cilta-cel. HLH / MAS can occur with CRS or other neurotoxicities. Prolonged and / or recurrent cytopenias with bleeding and infection and requirement or stem cell transplantation for hematopoietic recovery have occurred following treatment with cilta-cel.

[0246] Treatment Plan

[0247] All consented participants entered the screening phase for up to 28 days from consent date and eligibility was determined within this window. Once determined eligible, participants were cleared for apheresis to be completed within 40 days from the date of eligibility determination, except as noted in the relevant Schedule of Events. See Tables 7-9.

[0248] Eligible participants underwent apheresis for collection of peripheral blood mononuclear cells (PBMC). Study enrollment was defined at the day of apheresis. Cilta-cel was generated from T-cells selected from the apheresis. Participants for whom apheresis or manufacturing fails were allowed a second attempt at apheresis.

[0249] Bridging therapy (anti-plasma cell directed treatment between apheresis and the first dose of the conditioning regimen) was not needed while waiting for manufacturing of cilta-cel.

[0250] After notification of successful manufacturing of cilta-cel, participants received a lymphodepleting conditioning regimen of fludarabine and cyclophosphamide. Once cilta-cel had been received, participants received their cilta-cel infusion. Conditioning regimen was administered any day between day -7 and -5 and continued for 3 consecutive days. Cilta-cel infusion took place on Day 1.

[0251] A safety run-in of cilta-cel treatment was performed as follows. There were 3 participants each in two safety run-in phases who were observed for dose-limiting toxicities for 60 days. Cilta-cel infusion of participants within each safety run-in cohort was staggered by 6 weeks.

[0252] Dose-Limiting Toxicities (DLTs) were defined as follows: Grade 4 non-hematologic toxicity of any duration including Grade 4 CRS and ICANS; Grade 3 CRS that does not improve to a grade 2 or less in 72 hours following adequate therapy; Grade 3 neurological toxicity of any duration; Grade 3 toxicity of any duration involving vital organs (cardiac, pulmonary) - exceptions could be made if associated with CRS; other Grade 3 toxicity lasting > 72 hours - exceptions could be made for Grade 3 abnormal hepatic or renal function tests that improve to grade 2 or less within 7 days; Grade 3 hypersensitivity reaction that was not reversible to Grade 2 or less within 24 hours; and Grade 4 neutropenia or thrombocytopenia lasting more than 28 days.

[0253] Dose / Cohort Levels for the Safety Run-in were as follows:Dose Level Dose0 0.75xl06CAR-positive viable T-cells / kg-1 0.5xl06CAR-positive viable T-cells / kg-2 0.3xl06CAR-positive viable T-cells / kg

[0254] In COHORT ONE of the safety run-in phase, 3 participants were treated 6 weeks apart at a dose of 0.5xl06CAR-positive viable T-cells / kg (Dose Level -1) and each participant was observed for 60 days post-treatment. Further treatment was held until FDA had reviewed the adverse event data of the first 3 participants in the safety run-in cohort and approved treatment of next 3 safety run-in participants.

[0255] To achieve the cell dose at either Dose Levels -1 or -2, cilta-cel product was thawed and dose-modified at the clinical site within an ISO-5 biosafety cabinet following aseptic processing standard operating procedures. Dose calculations were verified by the investigational new drug holder based on data points provided by the investigator’s product COA.

[0256] The following were possible outcomes from COHORT ONE. If 0 out of 3 participants had DLTs as listed above, then 3 participants would be treated in COHORT TWO at target dose level 0.75xl06CAR-positive viable T-cells / kg. Participants at Dose Level 0 would receive thawed cilta-cel product unmanipulated with the entire product infused. Product delivered was at manufacturer’s current specifications of 0.5-1.0xl06CAR-positive viable T cells / kg. Actual dose level infused was recorded based on information provided on product COA. If 1 out of 3 participants had DLTs as listed above, then another 3 patients were treated within COHORT ONE / Dose Level -1 at the same dose level (i.e., dose 0.5xl06CAR-positive viable T-cells / kg) and observed each for 60 days and follow the COHORT ONE decision plan. If 2 or more participants had DLTs as listed above, then the trial would be held, and the dose would be modified to a dose of 0.3xl06CAR-positive viable T-cells / kg or cytoreduction therapy would be considered before cilta-cel infusion.

[0257] Once clear, COHORT TWO of 3 participants at a target dose of 0.75xl06CARpositive viable T-cells / kg were treated and each participant was observed for 60 days posttreatment. further treatment was held until FDA had reviewed the adverse event data of the first 3 participants in the safety run-in cohort and approved further treatment in the expansion cohort. Of note, additional participants could undergo screening assessments and apheresis for the expansion cohort while FDA approval to proceed with treating patients in the expansion cohort is pending.

[0258] Toxicity data was reviewed with the FDA with the following possible outcomes. If 0 out of 3 participants had DLTs as listed above, then the remaining expansion cohort participants were enrolled at target dose level 0.75xl06CAR-positive viable T-cells / kg. If 1 out of 3 participants had DLTs as listed above, then another 3 participants were treated in COHORT TWO safety run-in at the same target dose level (i.e., 0.75xl06CAR-positive viable T-cells / kg). Each participant for would be observed for 60 days following treatment, following the COHORT TWO decision plan. If 2 out of 3 participants had DLTs as listed above, then the trial was held, and the protocol was modified based on guidance from the FDA and investigators to either modify the dose of CAR-T to a dose of 0.5xl06CAR-positive viable T-cells / kg or cytoreduction therapy was considered before cilta-cel infusion.

[0259] Toxicity was continuously monitored from the time a signed and dated informed consent was obtained until 100 days after infusion of cilta-cel regardless of if progressive disease (PD) occurred prior to Day 100 or subsequent anti -myeloma therapy was started prior to Day 100, and 100 days after infusion of cilta-cel. Beyond the adverse event reporting period,only severe adverse events (SAEs), regardless of causality and non-serious adverse events (AEs) that were considered related to a study drug, needed to be reported until the end of the study except as defined for delayed AEs. In addition, events of HBV reactivations were reported during the first-year post-infusion of cilta-cel.

[0260] Adverse events were followed by the investigator as specified below graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE Version 5.0), with the exception of CRS and ICANS. CRS was evaluated according to the American Society for Blood and Bone Marrow Transplantation (ASBMT) consensus grading. ICANS was graded using the ASBMT (American Society for Transplantation and Cellular Therapy [ASTCT]) consensus grading. In addition to capturing ICANS and CRS adverse events (graded by ASTCT consensus grading), all individual symptoms of CRS (e.g., fever, hypotension) and CAR-T cell-related neurotoxicity (e.g., depressed level of consciousness, seizures) were captured as individual adverse events and graded by CTCAE criteria. Neurotoxicity that was not temporally associated with CRS, or any other neurologic adverse events that did not qualify as ICANS, was graded by CTCAE criteria. Events of neurotoxicity or exacerbation of existing neurologic adverse events was reported for duration of the study post infusion of cilta-cel.

[0261] Expected toxicities and potential risks for all agents are described below. No investigational or commercial agents or therapies other than those described below were administered with the intent to treat the patient’s smoldering multiple myeloma.

[0262] Apheresis

[0263] Participants must have had no evidence of a clinically significant infection prior to apheresis. Eligible participants underwent leukapheresis for approximately 9-14L blood volume to target 9xl09Total White Blood Cells (WBC), (Acceptable range: 6 to 12xl09Total WBCs).

[0264] Mononuclear cell collection (MNC), Apheresis product was transferred and placed into a 2-8°C shipper within one hour (60 minutes) of the end of collection. If more time was required prior to the transfer, the apheresis product was stored in a monitored refrigerator or intermediary transport at 2-8°C until the time of shipment occurs.

[0265] If the relevant criteria were not met, the apheresis was delayed until all criteria were satisfied.

[0266] The investigator was contacted if evidence of rapid disease progression was observed between screening and apheresis. Subjects met the following criteria to proceed withapheresis: clinical laboratory values required for enrollment resulted within 24-hours prior to apheresis; negative pregnancy test for women of childbearing potential up to 72 hours prior to apheresis; no antitumor therapy; cumulative dose of corticosteroids not exceeding equivalent to >140 mg prednisone within the 14 days prior to apheresis; no evidence of serious active viral, bacterial, or uncontrolled systemic fungal infection - subjects on anti -infective agents within 7 days prior to apheresis must receive approval to proceed from the sponsorinvestigator; no major surgery < 2 weeks prior to apheresis; no live, attenuated vaccines < 4 weeks prior to apheresis; no supplemental oxygen use to maintain adequate oxygenation; and no new arrhythmia or other cardiac adverse events unless controlled with medical management and approved by the medical monitor.

[0267] For subjects who required a repeat apheresis, the following screening assessments were collected before the second apheresis: weight, hematology laboratory assessments, chemistry laboratory assessments, and echocardiogram or MUGA (if clinically indicated). If the second apheresis fell outside of the 28-day window, all screening assessments (except bone marrow collection) must be repeated).

[0268] Stem Cell Mobilization

[0269] All participants who were able or willing to pursue stem cell transplantation underwent stem cell mobilization and collection at the investigator’s discretion as part of standard of care practice as shown in the schema of the protocol (FIG. 7) and before CAR-T infusion. Participants underwent stem cell collection after their T-cell apheresis and during manufacturing period prior to their scheduled lymphodepletion and CAR T-cell infusion. Those who forwent mobilization and collection did not receive any difference in care or therapy than those who choose to pursue mobilization and collection. Stem cell mobilization was performed with fdgrastim alone or filgrastim with plerixafor per institutional standard. In participants that were unable to achieve adequate stem cell yield with filgrastim and plerixafor could be used per institutional standard.

[0270] Only serious adverse events and study drug related adverse events were captured during the time of mobilization and subsequent recovery, as this was considered part of standard of care procedures.

[0271] Lvmphodepleting Conditioning Regimen

[0272] After notification that cilta-cel had been successfully manufactured and patient met safety criteria for treatment, subjects were administered a conditioning regimen of IV cyclophosphamide 300 mg / m2and fludarabine 30 mg / m2daily for 3 consecutive days to startany day between day -7 to -5. Cilta-cel was administered at a total targeted dose as described above 5 to 7 days after start of the conditioning regimen.

[0273] Cyclophosphamide is a nitrogen mustard-derivative that acts as an alkylating agent following conversion to active metabolites in the liver and has potent immunosuppressive activity. The serum half-life after IV administration ranges from 3 to 12 hours; the drug and / or its metabolites can be detected in the serum for up to 72 hours after administration.

[0274] Fludarabine phosphate (hereafter, fludarabine) is a synthetic purine nucleoside that differs from physiologic nucleosides in that the sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist antimetabolite.

[0275] The conditioning regimen of cyclophosphamide 300 mg / m2and fludarabine 30 mg / m2daily for 3 doses leads to lymphodepletion and helps to promote CAR-T cell expansion in the patient.

[0276] Participants must have had no evidence of a clinically significant infection prior to initiation of conditioning regimen. After criteria in were met, participants received lymphodepleting conditioning regimen. Participants began the conditioning regimen 5 to 7 days prior to planned cilta-cel infusion.

[0277] If the relevant criteria were not met, the conditioning regimen was delayed until all criteria were satisfied.

[0278] The 3 -day conditioning regimen of cyclophosphamide and fludarabine was administered in accordance with the below daily dosing instructions. IV hydration with a balanced crystalloid was administered according to institutional guidelines prior to cyclophosphamide on the day of infusion followed by cyclophosphamide 300 mg / m2IV over 30 minutes followed by fludarabine 30 mg / m2IV over 30 minutes followed by additional IV hydration with a balanced crystalloid according to institutional guidelines at the completion of the cyclophosphamide infusion. Any pre-medications given prior to initiating lymphodepleting chemotherapy were per investigator discretion. Subjects were instructed to drink plenty of liquids during and for 24 hours following the chemotherapy (approximately 2 liters / 24 hours). In general subjects were kept well-hydrated but closely monitored to prevent fluid overload.

[0279] Subjects had to meet the following criteria to proceed with cyclophosphamide and fludarabine dosing. Subject met clinical laboratory values required for enrollment, with the following exception: lymphocyte count of >0.3 x 109 / L. Transfusion support was permitted to maintain a hemoglobin of >8.0 g / dl (>5mmol / L) as needed, and platelets of >50 x 109 / L until 3 days before the hematology laboratory test, preceding lymphodepletion. Myeloid growthfactors were permited up to 1 day prior to the start of the conditioning regimen. Pegylated myeloid growth factors (i.e., pegfdgrastim) were prohibited. Subject must have had an ECOG performance status grade of 0 or 1. Subjects must have had a negative pregnancy test for women of childbearing potential up to 72 hours prior to the first dose of the conditioning regimen. Subject must not have received antitumor therapy. Subjects must have had no signs of active infection. For subjects requiring systemic anti-microbial treatment or with temperature >38.0 degrees Celsius within 7 days prior to the first dose of conditioning regimen, the investigator must have received approval to proceed from the sponsor-investigator. Subjects may not have had major surgery within 2 weeks prior to conditioning regimen dosing. Subjects may not have had live, atenuated vaccines within 4 weeks prior to conditioning regimen dosing. Subjects may not have required supplemental oxygen use to maintain adequate oxygenation. Subjects must not have had new arrhythmia or other cardiac adverse events unless controlled with medical management and approved by the medical monitor. Subjects were evaluated for the presence of an indwelling catheter prior to the first dose of the conditioning regimen.

[0280] Cilta-cel Infusion

[0281] Cilta-cel is a subject-specific product. Cilta-cel was administered at a targeted infused dose for the overall study of 0.75 x 106CAR-positive viable T-cells / kg (with a maximum total dose of 1 x 108CAR-positive viable T-cells); the cohort to which patient was enrolled dictated the dose of the individual patient, i.e., dose 0.5 x 106for Cohort 1 / Dose Level -1 and target of 0.75 x 106for Cohort 2 / Dose Level 0.

[0282] Participants must have had no evidence of a clinically significant infection prior to cilta-cel infusion. After criteria in were met, participants received their infusion of cilta-cel. Participants began the conditioning regimen 5 to 7 days prior to planned cilta-cel infusion.

[0283] All participants were hospitalized to receive treatment with cilta-cel followed by an observation period. Participants remained hospitalized for approximately 2 weeks post-cilta-cel infusion. Participants were not discharged from the hospital until all cilta-cel-related non-hematological toxicities returned to < Grade 1 or baseline. Participants were discharged with non-critical and clinically stable or improving toxicities, even if > Grade 1, if deemed appropriate by the investigator. Participants remained hospitalized for ongoing cilta-cel-related fever, hypotension, hypoxia, or ongoing central neurological events > Grade 1, or if deemed necessary by the investigator.

[0284] Given the possibility that a participant could develop CRS or a neurologic event after discharge from the hospital, participants were required to stay within 2 hours of the hospital and in the care of a competent adult for 28 days following cilta-cel infusion. Participants were required to document their temperature at least twice daily for 60 days following cilta-cel infusion. Participants were educated on potential symptoms such as fever, dyspnea, confusion, aphasia, dysphasia, somnolence, encephalopathy, ataxia, and tremor. If participants developed these symptoms, they were instructed to immediately contact the principal investigator and / or seek immediate medical attention.

[0285] Subjects were evaluated for safety on the day of cilta-cel infusion. If a significant health status change (e.g., clinical deterioration, rapidly progressing disease) occurred following the start of the conditioning regimen and prior to cilta-cel administration, the investigator was contacted prior to dosing.

[0286] Infusion of cilta-cel was delayed if any of the following events occurred. The subject showed signs of active infection. For subjects requiring systemic anti-microbial treatment, or with temperature >38.0 degrees Celsius within 48 hours before cilta-cel infusion, investigator was consulted prior to dosing. Subjects with Grade 3 non-hematologic toxicities of cyclophosphamide and fludarabine conditioning (except for Grade 3 nausea, vomiting, diarrhea, or constipation). If resolution of these events to Grade <1 took more than 14 days, the conditioning regimen was re-administered (cyclophosphamide 300 mg / m2and fludarabine 30 mg / m2daily for 3 days) after a minimum of 21 days following the first dose of the first conditioning regimen (cyclophosphamide and fludarabine).

[0287] Cilta-cel was administered as summarized in Table 10.

[0288] Approximately 4 weeks after apheresis, and after the site is notified that manufacture and quality testing of cilta-cel had been completed, each subject received a conditioning regimen of intravenous (IV) cyclophosphamide 300 mg / m2and fludarabine 30 mg / m2at 3 daily doses; sponsor-investigator approval was obtained to change the conditioning regimen schedule. Cilta-cel was administered as a single infusion 5 to 7 days after the start of the conditioning regimen (the first day of conditioning is any day between Day -7 to Day -5, and the day of cilta-cel infusion is Day 1). Cyclophosphamide and fludarabine were administered using administration procedures and supportive care according to the site’s standard of care. Cilta-cel was administered as described in the investigational product preparation instructions (IPPI).

[0289] A strategy of staggered dosing with cilta-cel was applied, whereby the preceding patient must have been at least 6 weeks apart before the subsequent patient could be treated. There was a 60-day DLT period between cohorts for the first 6 subjects.

[0290] In the event a cilta-cel product that did not meet pre-specified release criteria was produced during the manufacturing procedures, the investigator evaluated the risk / benefit for administration of the affected product and determined if the supply of the product to the treating physician could be considered. If required, approval from the relevant health authorities for use of the product was obtained. In the event the supply of the affected product was deemed appropriate, the investigator informed the study subject that the product did not meet release specifications prior to administration.

[0291] Prior to cilta-cel infusion, subjects received premedication as noted in Table 12. Corticosteroids were not used during pre-infiision.

[0292] Definition of Dose-Limiting Toxicity (DLT)

[0293] The dose de-escalation evaluation period was defined as 60 days after the infusion of cilta-cel. Toxicities that were considered at least possibly related to cilta-cel and that occur during the dose de-escalation evaluation period were considered for dose limiting toxicity (DLT) assessment.

[0294] The Dose Limiting Toxicity Criteria are as follows: Grade 4 non-hematologic toxicity of any duration including Grade 4 CRS and ICANS; Grade 3 CRS that did not improve to a Grade 2 or less in 72 hours following adequate therapy; Grade 3 neurological toxicity of any duration; Grade 3 toxicity of any duration involving vital organs (cardiac, pulmonary). Exceptions could be made if associated with CRS; Other Grade 3 toxicity lasting > 72 hours. Exceptions could be made for Grade 3 abnormal hepatic or renal function tests that improve to grade 2 or less within 7 days; Grade 3 hypersensitivity reaction that was not reversible to Grade 2 or less within 24 hours; and Grade 4 neutropenia or thrombocytopenia lasting more than 28 days.

[0295] If more than 1 out of the first 3 subjects at any dose level met DLT criteria during the 60-day evaluation period, then the additional 3 participants were enrolled at the same dose level and the toxicity was reviewed with the FDA and patients were monitored using the cohort specific criteria.

[0296] Adverse events (AEs) were evaluated according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE Version 5.0), with the exception of CRS and CAR-T cell-related neurotoxicity (e.g., ICANS). CRS was evaluatedaccording to the ASBMT consensus grading. CAR-T cell-related neurotoxicity (e.g., ICANS) was graded using the ASBMT consensus grading. Assessments for ICE was performed as specified in the Schedule of Events (Tables 7 and 9) in both the safety run-in and expansion portions of the study.

[0297] General Concomitant Medication. Supportive Care Guidelines, Permitted Medications, and Prohibited Therapies

[0298] Throughout the study, investigators could prescribe concomitant medications or treatments deemed necessary to provide adequate supportive care. All medications (including prescription and over-the-counter products, and transfusions of blood products) different from the study treatment were recorded throughout the study beginning with the signing of the informed consent form (ICF) until at least 100 days after infusion of cilta-cel or until the start of subsequent systemic anticancer treatment, if earlier. After 100 days, only adverse events that were considered related to study drug needed to be reported until the end of the study. This included concomitant therapy and any medication used to treat or support adverse events or serious adverse events (within or beyond 100 days after infusion). Recorded information included a description of the type of the drug, dosing regimen, route of administration, duration of treatment, and its indication. All medications, including details of previous anticancer treatment, were documented in the appropriate section of the eCRF.

[0299] Investigators also reported 1) all medications given to prevent or treat COVID-19 up to 1-year post-cilta-cel infusion, and 2) all cases of COVID-19, regardless of grade (including asymptomatic COVID- 19) up to 1 year from cilta-cel infusion.

[0300] Prior to cilta-cel infusion, subjects received premedication as noted above. Corticosteroids were not used during pre-infusion.

[0301] The following are examples of supportive therapies that could be used during the study. Standard supportive care therapies (antiemetics, antidiarrheals, anticholinergics, antispasmodics, antipyretics, antihistamines, analgesics, antibiotics and other antimicrobials, histamine receptor [H2] antagonists or proton pump inhibitors, and other medications intended to treat symptoms or signs of disease) and therapies intended to treat CAR-T cell-related toxicity (i.e., CRS) as clinically indicated, according to institutional standards and as deemed necessary by the investigator. Bisphosphonates could be initiated (if not already being administered) unless contraindicated at least 60 days after cilta-cel infusion and continued until disease progression was established or for at least 2 years. In the case of severe adverse events such as hypercalcemia, bisphosphonates may be administrated as clinically indicated,according to institutional standards and as deemed necessary by the investigator. Transfusion support was permitted to maintain a hemoglobin of >8.0 g / dL (>5mmol / L) as needed, and platelets of >50 x 109 / L until 3 days before the hematology laboratory test, preceding lymphodepletion. Myeloid growth factors were permitted up to 1 day prior to the start of the conditioning regimen. Pegylated myeloid growth factors (i.e., pegfilgrastim) were prohibited. Documented infectious complications were treated with oral or IV antibiotics or other anti-infective agents as considered appropriate by the treating investigator, according to standard institutional practice. Chemotherapy agents used to treat CAR-T cell -related toxicities were permitted upon consultation with the investigator.

[0302] The following medications were prohibited during the study. The investigator was notified in advance (or as soon as possible thereafter) of any instances in which prohibited therapies were (to be) administered. Corticosteroid use was avoided, except for the treatment of CRS or CAR-T cell-related neurotoxicity (e.g., ICANS). Alternative therapies, if feasible, were given prior to corticosteroids. Any chemotherapy, anticancer immunotherapy (other than cilta-cel), or experimental therapy, or protocol- specific therapies which may be used in conjunction with cilta-cel, were prohibited. While in follow-up, emergency orthopedic surgery or radiotherapy was generally prohibited, but could be allowed in the absence of disease progression. Cases were discussed and approved by the investigator. Such emergency radiotherapy could consist of localized radiotherapy for pain control or for stabilization of an extensive bone lesion at high risk of pathologic fracture or damage to surrounding tissues. Nonsteroidal anti-inflammatory agents was avoided to minimize the risk of exacerbation of potential sub-clinical myeloma-related kidney disease. Based on the investigator’s clinical judgement, low-dose aspirin could be continued for thromboprophylaxis. Other immunosuppressant agents unless used as protocol-specified pre- or post-treatment medications to treat an adverse event (e.g., CRS) were prohibited. Vaccination with live, attenuated vaccine after signing consent and in the < 4 weeks prior to the infusion of cilta-cel, and for 100 days after infusion of cilta-cel were prohibited. The use of IV contrast infusions should be avoided to prevent myeloma-related kidney disease. If administration of IV contrast is necessary, then adequate precautions including hydration are indicated. Pegylated myeloid growth factors (i.e., pegfilgrastim) were prohibited within the first 100 days after infusion of cilta-cel.

[0303] Criteria for Taking a Participant Off Protocol Therapy

[0304] Duration of therapy depended on individual response, evidence of disease progression and tolerance. In the absence of treatment delays due to adverse event(s), treatment could continue until one of the following criteria applied: the investigator believed that for safety reasons or tolerability reasons (e.g., adverse event) it was in the best interest of the subject to discontinue study treatment; Grade 3 nonhematologic toxicity related to cyclophosphamide and fludarabine occurred and precluded retreatment with cyclophosphamide and fludarabine prior to cilta-cel infusion; the subject received concurrent (non-protocol) anticancer treatment; confirmed disease progression per IMWG criteria between the time of conditioning therapy and infusion of cilta-cel; the participant decided to withdraw from the protocol therapy; and non-compliance with study treatment or procedure requirements.

[0305] The primary reason for treatment discontinuation was documented, and the subject was followed per standard of care until recovery from cyclophosphamide and fludarabine conditioning regimen. If a subject's study treatment was discontinued for any reason, this did not result in automatic withdrawal of the subject from the study.

[0306] Follow-Up

[0307] Participants who completed treatment or who discontinued therapy for any reason other than withdrawal of consent, death, or development of CRAB / MDE will be monitored every three months (± 2 weeks) for the first 5 years (Active Follow-up Phase) and then every 6 months thereafter until new SMM / MM related therapy, death, or withdrawal of consent, whichever occurrs first, up to 15 years (Long-term Follow-up Phase). Further details can be found in Table 8 (Schedule of Events for Follow-up Procedures / Assessments After Treatment with cilta-cel).

[0308] During the active follow-up period, physical exams were voluntary, and virtual visits or telehealth visits were acceptable. Serum Protein Electrophoresis (SPEP) and Immunofixation were conducted, with mass spectrometry performed as feasible. Serum Free Light Chains (SFLC) tests were required, and Urine Protein Electrophoresis (UPEP) and Immunofixation were necessary only for those where disease is tracked by the urine. Serum Immunoglobulin tests were also conducted. PET / CT assessments were strongly recommended yearly and at the time of suspected progression, with the same assessment modality used consistently across the duration of the trial. Bone marrow biopsies are required at several time points during the first year and are strongly recommended at Month 18 and Year 2 post end of treatment (EOT), yearly until the patient is off-study, and at the time of suspected progression.Collection schedules are provided for in Table 9 for the collection of blood and bone marrow aspirate during the active follow-up phase.

[0309] Following confirmed CRAB / MDE, or completion of the active follow-up phase, participants were followed for overall survival status, second primary malignancies, first subsequent therapy, and best response, and second therapy and best response. Survival follow up was completed by phone contact, email or other method not mentioned here. Death information from public sources, (e.g., death registry, obituary listing, etc.), could also be used when it is available and verifiable. The date and regimen of the first subsequent therapy was recorded in the eCRF if it occurred during overall survival follow up. Survival follow up will occur at minimum every 6 months until death, withdrawal of consent or second progression on a myeloma directed therapy, up to 15 years.

[0310] Criteria for Taking a Participant Off Study

[0311] A subject was withdrawn from the study for any of the following reasons: lost to follow-up; withdrawal of consent; failure to manufacture cilta-cel after 2 apheresis attempts; the investigator discontinued the study; or death. If a subject was lost to follow-up, every reasonable effort must have been made by the study-site personnel to contact the subject and determine the reason for discontinuation / withdrawal. If a subject withdrew consent following dosing with cilta-cel, study assessments for the last visit in the post-infusion period were completed prior to withdrawal of consent, if feasible. The reason for taking a participant off study, and the date the participant was removed, were documented in the case report form (CRF).

[0312] Management Guidelines for Potential Risks

[0313] Potential safety risks are based on the known mechanism of action of cilta-cel. Special attention was given to immunological effects due to release of cytokines and toxicities associated with depletion of cells that express BCMA (e.g., plasma cells).

[0314] By stimulating an inflammatory cascade, there is toxicity in other tissues or organs by non-specific immune cell activation. Therefore, special attention was given to both immunological and immunogenicity-related toxicities. Potential safety risks and mitigation strategies are outlined in Table 13.

[0315] Cytokine Release Syndrome (CRS) is a toxicity shown to be associated with various CAR-T cell therapies in clinical studies of hematologic malignancies. The presence of CRS correlates with expansion and activation of CAR-T cells. CRS is associated with the elevation of cytokines, namely IL-2 receptor alpha, IL-6, and IL- 10. Clinical symptoms are associatedwith increased inflammatory markers, including C-reactive protein (CRP), ferritin, and IL-6. The onset of CRS typically occurs within the first week of treatment; however, in participants with B-cell malignancies, a delayed CRS occurs at the time of peak levels of CAR-T cells in blood and bone marrow. Participants were closely monitored for early signs and symptoms indicative of CRS, such as fever, and immediately hospitalized. Body temperature should be monitored twice daily for 60 days post CAR-T cell infusion.

[0316] Clinical symptoms indicative of CRS may include but are not limited to fever (with or without rigors), arthralgia, nausea, vomiting, tachycardia, hypotension, headache, confusion, tremor, and delirium, dyspnea, pulmonary edema, and capillary leak. Potentially lifethreatening complications of CRS may include cardiac dysfunction, adult respiratory distress syndrome, neurologic toxicity, renal and / or hepatic failure, and disseminated intravascular coagulation.

[0317] Infection and CRS may have a similar presentation. Therefore, investigators were strongly encouraged to evaluate for an infection at the first signs or symptoms of CRS were and imaging should be obtained; the clinical signs and symptoms determined which tests are appropriate.

[0318] Supportive care for CRS (including but not limited to anti-pyretic agents, IV fluid support, vasopressors, supplemental oxygen, etc.) was administered according to the clinical manifestations of the participant’s illness. Similarly, ancillary testing such as B-type natriuretic peptide (BNP) assessment, echocardiograms, arterial blood gas, assessments of coagulation laboratory tests, etc. were performed if clinically indicated.

[0319] Laboratory testing to monitor for disseminated intravascular coagulation, a manifestation of CRS, were performed in addition to daily monitoring of chemistry and hematology assessments (including ferritin and CRP) when fever or other signs of potential CRS are present. In addition, pulmonary, renal and hepatic function were monitored closely.

[0320] Rarely, severe CRS can evolve into a presentation consistent with hemophagocytic lymphohistiocytosis (HLH) / macrophage activation syndrome (MAS) that may require additional therapy. In these cases, laboratory testing may reveal high serum levels of ferritin, lactate dehydrogenase, triglycerides, soluble CD25 (also known as soluble IL-2 receptor alpha), and cytokines (such as fFNy and IL-6), and low serum levels of fibrinogen.

[0321] Trained clinical personnel were prepared to intervene in the event of CRS. Resources necessary for resuscitation (i.e., agents such as epinephrine and aerosolized bronchodilator; medical equipment such as oxygen, tracheostomy equipment, and a defibrillator) were readilyavailable. Tocilizumab was available prior to administration of cilta-cel. Vital signs and laboratory parameters were monitored at regular intervals until normal. Additional specimens for PK and pharmacodynamic testing were collected as outlined in the protocol.

[0322] Infection and CRS may have a similar presentation. Therefore, investigators were strongly encouraged to evaluate for an infection at the first signs or symptoms of CRS. Blood cultures and imaging were obtained: the clinical signs and symptoms determined which tests were appropriate. Recommendations for the clinical management of CRS are provided in Table 3.

[0323] At the first sign of CRS (such as fever) participants were immediately hospitalized for evaluation. The use of myeloid growth factors, particularly G-CSF, was avoided during CRS. Tocilizumab intervention was considered with presenting symptom of fever per investigator discretion when other sources of fever had been eliminated and early tocilizumab was considered in participants at high risk of severe CRS (including high baseline tumor burden, early fever onset, or persistent fever after 24 hours of symptomatic treatment). Other cytokine-targeting therapies (e.g., IL-1 and / or anti-tumor necrosis factor alpha) could be used based on institutional practice, especially for cases of CRS which do not respond to tocilizumab and corticosteroids. Therapy directed at reduction or elimination of CAR-T cells, including chemotherapy, was considered in consultation with the Sponsor Investigator for participants who developed high grade CRS with laboratory findings overlapping with HLH / MAS (including hyperferritinemia) that remained severe or life-threatening following prior therapies, including tocilizumab and corticosteroids. CRS was captured as an AE of special interest.

[0324] Based on the specific mode of action of cilta-cel, severe or serious neurologic toxicities, including CAR-T cell neurotoxicity, i.e., immune effector cell-associated neurotoxicity syndrome (ICANS) and other neurotoxicities, could occur. Participants were monitored for neurotoxicity until the end of the study.

[0325] All neurologic AEs, including CAR-T cell neurotoxicity (e.g., ICANS), were captured as AEs of special interest.

[0326] Participants had the ICE assessment tool performed within 24 hours prior to cilta-cel infusion and daily after the first symptoms of neurotoxicity were suspected and until resolution. The ICE Tool was performed more frequently until neurotoxicity symptoms resolved. Neuroimaging (e.g., magnetic resonance imaging [MRI]) was performed at screening and / or neurology consultation was provided if pre-existing disease was suspected.

[0327] Participants were monitored for neurologic toxicities, including but not limited to, headache, convulsions, speech disorders, visual disorders, disturbances in consciousness, confusion, disorientation, and coordination and balance disorders, or mental status changes. If these or other neurologic toxicities were observed, regardless of causality, the medical monitor was consulted. Hospitalization was required for Grade 2, 3, or 4 CAR-T cell neurotoxicity (e.g., ICANS) temporarily associated with CRS.

[0328] At the first sign of neurotoxicity, neurology consultation and evaluation were considered. Alternative etiologies including infectious etiologies (e.g., viral origin such as human herpes virus [HHV]-6, HHV-7) were ruled out if clinically indicated. For signs of seizures or raised intracranial pressure (ICP)Zcerebral edema, the participant was transferred to the intensive care unit and treated according to institutional guidelines or practices.

[0329] General management for ICANS is summarized in Table 11. Guidelines for the management of raised ICP / cerebral edema are summarized in Table 14.

[0330] If concurrent CRS was suspected during the neurologic toxicity event, the following was administered: corticosteroids according to the more aggressive intervention based on the CRS and neurologic toxicity grades in Table 3 and 11; Tocilizumab according to CRS grade in Table 3; antiseizure medication according to neurologic toxicity in Table 11.

[0331] Participants were monitored closely for other neurotoxicities with clinical presentation for the duration of the study after cilta-cel infusion. If any neurologic or psychiatric symptoms were noted, the medical monitor was contacted, and the participant was referred immediately to a neurologist for a full evaluation.

[0332] Particular attention was paid to the appearance of any of the following movement and neurocognitive treatment emergent adverse events (TEAEs), often with subtle onset: movement impairments (e.g., micrographia or changes in handwriting, tremors, bradykinesia, rigidity, shuffling gait, impaired balance and coordination, difficulty writing, difficulty performing activities of daily living like dressing or feeding oneself); cognitive impairments (e.g., memory loss or forgetfulness, disturbances in attention, mental slowness or fogginess, difficulty speaking or slurred speech, difficulty reading or understanding words); and personality change (e.g., reduced facial expression, flat affect, reduced ability to express emotions, less communicative, disinterest in activities).

[0333] Other neurotoxicities characterized by movement and neurocognitive TEAEs were observed at a higher frequency in participants with high burden of disease and high CAR-Tcell expansion and persistence, and in participants experiencing higher grade CRS (Grade 2 and above) and any grade ICANS.

[0334] Additional monitoring and mitigation strategies included enhanced bridging therapy to reduce baseline tumor burden, early aggressive treatment of CRS and ICANS, handwriting assessments for early detection of neurotoxicity symptoms, and extended monitoring and reporting time for neurotoxicity for the duration of the study after cilta-cel infusion.

[0335] Early detection, workup, and intervention was important to prevent neurologic toxicity from worsening. The following is a list of potential diagnostics that were considered in participants with new neurologic symptoms: positron emission tomography / computed tomography of the brain and / or brain MRI with perfusion and an electroencephalogram; lumbar puncture to rule out infection (in particular, John Cunningham virus, herpes zoster virus [HZV], herpes simplex virus-1 / 2, HHV-6, HHV-7, Epstein-Barr virus, CMV); serologic testing for HHV-6 and HHV-7 by polymerase chain reaction (PCR) for viremia; CSF flow cytometry and cytology were considered to rule out leptomeningeal disease; CSF analysis was considered to rule out paraneoplastic syndromes; and thiamine level (consider empiric thiamine replacement while awaiting results).

[0336] Therapy directed at reduction or elimination of CAR-T cells, including chemotherapy, was considered in consultation with the Sponsor Investigator for participants who developed neurotoxicity that remains unresponsive to other interventions.

[0337] Participants could exhibit cytopenias for several weeks following lymphodepleting chemotherapy and cilta-cel infusion. Severe thrombocytopenia could increase the risk of bleeding. Prolonged neutropenia could increase the risk of infection. Blood counts (evaluation of hematological parameters) were frequently monitored after cilta-cel infusion. Supportive care (e.g. , irradiated packed red blood cells and platelets, G-CSF for neutropenia) was provided based on institutional standards. The use of myeloid growth factors, particularly G-CSF, was avoided during CRS. Pegylated myeloid growth factors (i.e., pegfilgrastim) were prohibited. Parvovirus B19 monitoring by PCR was considered in participants experiencing prolonged neutropenia or a decline in neutrophil counts following recovery.

[0338] Cilta-cel was not administered to participants with active infection. Administration of cilta-cel could increase the risk of infection due to cytopenias or hypogammaglobulinemia. Participants were monitored frequently for infection and should have blood cultures obtained, serum inflammatory markers (CRP) monitored, and / or empiric antibiotics administered per institutional standards. Immunocompromised participants are at risk for opportunisticinfections. Prophylactic use of antibiotics, antivirals, or antifungals was considered for high-risk participants. Extended use of antimicrobial therapies for at least 6 months or consistent with post ASCT consensus guidelines after cilta-cel dosing was recommended. Prophylaxis for HZV, CMV, or other HHV reactivation was recommended during study treatment as clinically indicated.

[0339] Screening for HBV, hepatitis C virus, and human immunodeficiency virus (HIV) was performed and monitored as clinically indicated, and treatment was initiated as appropriate. CMV serology was considered at baseline, and monitored with PCR testing as clinically indicated per institutional guidance. _HBV reactivation, in some cases resulting in fulminant hepatitis, hepatic failure and death, may occur in participants treated with drugs directed against B-cells such as cilta-cel. Hepatitis cases have been reported in participants who are hepatitis B surface antigen (HBsAg) positive, and also in participants who are HBsAg-negative but hepatitis B core antibody (anti-HBc) positive. Participants with positive anti-HBc and / or positive anti-HBs had an HBV-DNA quantification test. Participants with positive HBV-DNA were excluded. Participants with negative HBV-DNA could be enrolled; however, HBV-DNA and AST / ALT laboratories were performed every 3 months for the first 12 months after dosing. Prophylaxis for participants at high risk of HBV reactivation was recommended per institutional guidance.

[0340] The LV vector used to manufacture cilta-cel was derived from the HIV-1 genome and pseudo-typed with a VSV-G envelope protein in place of the HIV envelope protein. Treatment with cilta-cel could result in a false positive HIV test by some commercial tests. A safety feature notable of this vector is the non-essential HIV-1 sequences are not present at all in transfer vector and packaging constructs, making it unlikely to generate a fully wild-type HIV-1 virus. Quantitative PCR testing for VSV-G sequences was performed on each batch of cilta-cel and participant peripheral blood samples collected during study.

[0341] Multiple factors may place a person at increased risk for severe or fatal COVID-19 infection; however, it appears receiving cilta-cel may place participants at greater risk, particularly in the initial few months after cilta-cel infusion before the immune system can recover. For this reason, COVID-19 vaccination was recommended about 3 months after cilta-cel infusion, even if participants were vaccinated before cilta-cel administration. The Sponsor-Investigator may also prescribe other COVID- 19 medications to potentially prevent or reduce the risk of infection or severe infection. Participants were educated to notify the SponsorInvestigator if they were diagnosed with COVID-19 (even if they have no or only minor symptoms), or have been exposed to someone with COVID- 19 infection.

[0342] Tumor Lysis Syndrome (TLS) is a combination of metabolic derangements caused by the massive and abrupt release of cellular components into the blood after rapid lysis of malignant cells. These metabolites can overwhelm the body’s homeostatic mechanisms and cause hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia. Although TLS is uncommon in participants with multiple myeloma, 1 participant in the ongoing Phase 1 Legend-2 study experienced a fatal TLS and CRS.

[0343] Early recognition and monitoring of signs and symptoms of TLS, including identification of abnormal clinical and laboratory values, may lead to successful prevention of the serious clinical complications of the condition. Participants were monitored closely for symptoms of TLS.

[0344] Management of TLS, including hyperkalemia, hyperuricemia, hyperphosphatemia, and hypocalcemia, was recommended. It was also required that high-risk participants, i.e., those with a high tumor burden (>60% plasma cell infiltrate on the bone marrow biopsy or aspirate [whichever is higher] or a participant with multiple extramedullary disease sites or plasmacytomas), were treated prophylactically in accordance with local standards (e.g., extra hydration; diuretics; allopurinol; and primary or secondary uricosuric agents, as indicated).

[0345] The risk of second primary malignancy is theoretical due to viral insertion (DNA integration) of the LV vector used to make cilta-cel. Insertional mutagenesis may activate a cellular proto-oncogene or disrupt a tumor suppressor gene, leading to malignant transformation events (i.e., second primary malignancies). If a participant was diagnosed with a secondary primary malignancy, a sample of the tumor was collected. Subsequent analysis, involving the examination of DNA, RNA, or proteins, was conducted to explore the presence of LV elements. Additionally, other samples, including but not limited to blood, tissue, or additional tumor samples, were requested as clinically indicated.

[0346] The potential presence of RCL was evaluated from whole blood samples of subjects treated with cilta-cel. To monitor if RCL emerges from cilta-cel, whole blood from participants was evaluated using a quantitative PCR assay against the LV vesicular stomatitis virus-G gene at approximately 3 months, 6 months, and 12 months after cilta-cel infusion (as specified in the Schedule of Activities). Yearly review of medical history will generally be sufficient for the participant for up to 15 years after cilta-cel infusion in the present study (until end of study) or in a separate long-term follow-up study. If any post-infusion samples were positive, furtherRCL analysis and more extensive participant follow-up was undertaken. Additional samples could be collected triggered by events which may be relevant, but not limited, to RCL per clinical assessment as specified in the Schedule of Activities. In addition, a blood sample was collected for RCL evaluation at SPM onset.

[0347] Second primary malignancies were managed per institutional standards. Second primary malignancies were reported for the duration of the study, irrespective of when they occurred, and subsequently will be collected in a long-term follow-up study until 15 years post dosing of cilta-cel. Second primary malignancy was captured as an AE of special interest.

[0348] CAR-T cells target BCMA-expressing B-cells, resulting in disruption of normal B-cell maturation to plasma cells, subsequently resulting in hypogammaglobulinemia. However, previous chemotherapy can also contribute to decreased normal B-cells and the incidence of hypogammaglobulinemia pre-infusion. Hypogammaglobulinemia is ameliorated with intravenous gamma globulin infusion. Ig levels were monitored after treatment as detailed in the protocol and more frequently if clinically indicated. Treatment was administered according to local guidelines, including administration of Ig replacement and monitoring for infection. Participants with IgG <400 mg / dL or recurrent infections (including HBV reactivation) were considered for prophylactic intravenous or subcutaneous IgG as per institutional guidelines.

[0349] Vaccination with live virus vaccines was not recommended for at least 6 weeks prior to the start of lymphodepleting chemotherapy.

[0350] Allergic reactions may occur with the infusion of cilta-cel. Serious hypersensitivity reactions including anaphylaxis, may be due to DMSO, dextran 40, or residual kanamycin in cilta-cel. Anaphylaxis is a serious allergic reaction that can be fatal. It typically causes more than one of the following: an itchy rash, throat or tongue swelling, shortness of breath, vomiting, light-headedness, and low blood pressure. These symptoms typically present over a period of minutes to hours, especially if exposure is intravenously. Participants were treated urgently per institutional standards, avoiding corticosteroid use if possible. Participants received premedication prior to cilta-cel dosing as detailed in the protocol. Participants remained at study site for approximately 2 hours after infusion for monitoring of severe reaction. If not hospitalized, participants remained in the presence of a competent adult and within 2 hours of the hospital for days following cilta-cel infusion.

[0351] Stopping Rules for Safety and Treatment Pause

[0352] All participants were evaluable for toxicity from the time of therapy initiation.

[0353] A safety monitoring committee (SMC) was composed of medical oncologists, research nurses, pharmacists, and biostatisticians with direct experience in cancer clinical research. The SMC reviewed each protocol up to four times a year with the frequency determined by the outcome of previous reviews. Information to be provided to the committee could include, up to-date participant accrual, all grade 2 or higher unexpected adverse events that have been reported, summary of all deaths occurring within 30 days of intervention, any response information, audit results, and a summary provided by the study team.

[0354] If a stopping rule was triggered according to the criteria stated in the decision plan section for the first 3 patients and then next 3 patients enrolled to cilta-cel in this protocol, treatment was paused, and the SMC and FDA reviewed and monitored toxicity from this study to provide guidance regarding whether to continue treatment or terminate further accrual to the study.

[0355] Regardless of the stopping rules, after accrual of 6 participants on cilta-cel, the investigator reviewed toxicity data for all participants. If any deaths occurred on study that could not be directly related to disease progression or extraneous causes, enrollment was paused and the SMC reviewed and monitored toxicity from this study to provide guidance regarding whether to continue enrollment or terminate the study.

[0356] Adverse Events

[0357] The following list of reported and / or potential AEs and the characteristics of an observed AE determined whether the event required expedited reporting in addition to routine reporting.

[0358] Expected toxicities included an up-to-date list of risks included in the most recent informed consent document. For apheresis, more common adverse events include pain, bruising, and discomfort where the needles enter the veins; tingling in the lips or fingers, numbness, or a “vibrating” sensation caused by the anticoagulant used during the apheresis process, which may be managed by calcium administration; lightheadedness or dizziness while blood is being circulated through the apheresis machine; and a lower number of red blood cells that can cause tiredness and shortness of breath, potentially requiring a blood transfusion. Less common adverse events include a low number of platelets, which may require a blood or platelet transfusion; nausea, vomiting, chest tightness, and muscle cramps; and flushing or reddening of the skin. Very rare adverse events include clotting in the apheresis machine or in a participant, which may be life-threatening; allergic reactions, seizures, air emboli (air bubbles in blood veins or arteries), or abnormal heart rhythms; swelling of the hands and feet or fluidretention; low blood pressure, high blood pressure, or a slow pulse; and infection at the site where the needles enter the veins.

[0359] Risks associated with cyclophosphamide include likely adverse events with more than a 50% chance of occurrence, such as nausea, vomiting, loss of appetite, abdominal discomfort or pain, and diarrhea. Frequent adverse events, with a 10-50% chance of occurrence, include neutropenia, febrile neutropenia, thrombocytopenia, and anemia. Rare adverse events, with less than a 1% chance of occurrence, include infusion reactions and hemorrhagic cystitis.

[0360] For fludarabine, likely adverse events with more than a 50% chance of occurrence include fever, chills, fatigue, and / or weakness. Frequent adverse events, with a 10-50% chance of occurrence, include neutropenia, febrile neutropenia, thrombocytopenia, anemia, cough, nausea, vomiting, diarrhea, muscle aches and pains, inflammation of the digestive tract, and loss of appetite. Occasional adverse events, with a 1-10% chance of occurrence, include infections. Rare adverse events, with less than a 1% chance of occurrence, include hemolytic anemia, autoimmune thrombocytopenia, and tumor lysis syndrome (TLS).

[0361] Following cilta-cel infusion, very common adverse events with greater than a 20% chance of occurrence include neutropenia, thrombocytopenia, anemia, lymphopenia, coagulopathy, tachycardia, diarrhea, nausea, pyrexia, fatigue, pain, chills, peripheral edema, cytokine release syndrome (CRS), upper respiratory infection, dyspnea, hypoxia, transaminase elevation, hypocalcemia, hypophosphatemia, decreased appetite, hypokalemia, musculoskeletal pain, headache, encephalopathy, peripheral neuropathy, dizziness, motor dysfunction, immune effector cell-associated neurotoxicity syndrome (ICANS), cough, hypotension, and insomnia. Common adverse events, with less than a 20% chance of occurrence, include cardiac arrhythmias, abdominal pain, pulmonary edema / fluid retention, joint swelling, pain, hyperbilirubinemia, hypogammaglobulinemia, nasal congestion, sepsis (including septic shock), bacterial infections, viral infections (such as COVID- 19), C-reactive protein level increase, myalgia, confusion / mental impairment, somnolence / lethargy, memory impairment, sleep disorders, muscle spasms, muscular weakness, aphasia, tremor, ataxia, paresis, delirium, personality changes, and renal failure.

[0362] Serious Adverse Events (SAE) refer to any untoward medical occurrence that results in death, is life-threatening (meaning the patient was at risk of death at the time of the event, not hypothetically), requires inpatient hospitalization or prolongation of an existing hospitalization, results in persistent or significant disability or incapacity (defined as a substantial disruption of a person’s ability to conduct normal life functions), is a congenitalanomaly or birth defect, is a suspected transmission of any infectious agent via administration of a medicinal product, or is a medically important event. A medically important event may not result in death, be immediately life-threatening, or require hospitalization, but may be considered serious when, based on appropriate medical judgment, it may jeopardize the patient, require medical or surgical intervention to prevent one of the outcomes listed above, or involve suspected transmission via a medicinal product of an infectious agent. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse. Any organism, virus, or infectious particle (e.g., prion protein transmitting Transmissible Spongiform Encephalopathy), whether pathogenic or nonpathogenic, is considered an infectious agent. Note that death for any reason was reported as a serious adverse event.

[0363] The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 were utilized for AE reporting.

[0364] For expedited reporting purposes only, adverse events for the agent(s) listed above were reported only if the adverse event varies in nature, intensity, or frequency from the expected toxicity information provided.

[0365] The attribution of the adverse event were categorized as follows: definite (the AE was clearly related to the study treatment), probable (the AE was likely related to the study treatment), possible (the AE might have been related to the study treatment), unlikely (the AE was doubtfully related to the study treatment), and unrelated (the AE was clearly not related to the study treatment).

[0366] In the event of an unanticipated problem or life-threatening complications treating, investigators were to immediately notify the investigator.

[0367] Investigators reported to the investigator any adverse event (AE) that occurred after the initial dose of study treatment, during treatment, or within 100 days of the last dose of treatment on the local institutional AE form.

[0368] Grade 1 lab abnormalities that were truly transient (i.e., not lasting for more than 48 hours) and not clinically significant (i.e., no treatment is indicated) did not need to be captured and did not need to be reported as they do not constitute a reportable adverse event.

[0369] Table 15 indicates which events were reported to the investigator.

[0370] Pregnancy

[0371] Any pregnancy that occurred in a female participant or a female partner of a male study participant were reported to the investigaotr. Pregnancies and suspected pregnancies (including a positive pregnancy test regardless of age or disease state) of a female subject occurring while the subject was on this study were considered immediately reportable events. The female subject was referred to an obstetrician / gynecologist, preferably one experienced in reproductive toxicity for further evaluation and counseling.

[0372] All neonatal deaths that occurred within 28 days of birth were reported, without regard to causality, as SAEs. In addition, any infant death after 28 days that the Investigator was is related to the in-utero exposure to any drugs should were reported to the investigator.

[0373] If a female partner of a male subject receiving investigational product became pregnant, the male subject taking the study medication notified the investigator, and the pregnant female partner was advised to call their healthcare provider immediately.

[0374] All initial reports of pregnancy in female subjects or partners of male subjects were reported by the study-site personnel within 24 hours of their knowledge of the event using appropriate pregnancy notification form. Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, stillbirth, congenital anomalies, and ectopic pregnancy) were considered serious adverse events and must be reported using the Serious Adverse Event Form. Any subject who became pregnant during the study discontinued further study treatment. Because the effect of the study treatment on sperm is unknown, pregnancies in partners of male subjects included in the study were reported as noted above. Follow-up information regarding the outcome of the pregnancy and any postnatal sequelae in the infant was required.

[0375] Special Reporting Situations

[0376] Safety events of interest that required expedited reporting or safety evaluation included, but are not limited to: overdose of a study treatment; suspected abuse / misuse of a study treatment; accidental or occupational exposure to a study treatment; medication error involving an investigational product (with or without subject / subject exposure to the study treatment, e.g., name confusion); exposure to a study treatment from breastfeeding; and any new incidence of Grade 3 or higher infections (including COVID- 19), irrespective of seriousness or causality.

[0377] SAEs (regardless of causality) were reported for the duration of the study postinfusion of cilta-cel and subsequently will be collected yearly in long-term follow-up study for up to 15 years post-infusion of cilta-cel.

[0378] Events that required an escalation of care when the participant is already hospitalized were recorded as an SAE. Examples of such events include movement from routine care in the hospital to the intensive care unit or if that event resulted in the prolongation of the existing planned hospitalization.

[0379] All serious adverse events that had not resolved by the end of the study, or that had not resolved upon discontinuation of the subject's participation in the study, were followed until any of the following occurs: the event resolves; the event stabilizes; the event returns to baseline, if a baseline value / status is available; the event can be attributed to agents other than the study treatment or to factors unrelated to study conduct; or it becomes unlikely that any additional information can be obtained (subject or health care practitioner refusal to provide additional information, lost to follow-up after demonstration of due diligence with follow-up efforts).

[0380] Suspected transmission of an infectious agent by a medicinal product were reported as a serious adverse event. Any event requiring hospitalization (or prolongation of hospitalization) that occurred during the course of a subject's participation in a study were reported as a serious adverse event, except hospitalizations for the following: routine monitoring hospitalizations post-infusion required per protocol; hospitalizations not intended to treat an acute illness or adverse event (e.g., social reasons such as pending placement in long-term care facility); surgery or procedure planned before entry into the study (note: hospitalizations that were planned before the signing of the ICF, and where the underlying condition for which the hospitalization was planned has not worsened, were not be considered serious adverse events; any adverse event that resulted in a prolongation of the originally planned hospitalization was reported as a new serious adverse event); or the administration of blood or platelet transfusions (hospitalization or prolonged hospitalization for a complication of such transfusion remains a reportable serious adverse event).

[0381] For reports of hospitalization, it is the sign, symptom or diagnosis which led to the hospitalization that is the serious event for which details were provided. Any event requiring hospitalization or prolongation of hospitalization that occurred during the study were reported as a serious adverse event, except hospitalizations for the following: hospitalizations not intended to treat an acute illness or adverse event (e.g., social reasons such as pending placement in long-term care facility); surgery or procedure planned before entry into the study. Hospitalizations that were planned before the start of data collection and where the underlying condition for which the hospitalization was planned has not worsened will not be consideredserious adverse events. Any adverse event that results in a prolongation of the originally planned hospitalization is to be reported as a new serious adverse event.

[0382] Disease progression was not recorded as an adverse event or serious adverse event term; instead, signs and symptoms of clinical sequelae resulting from disease progression / lack of efficacy were reported if they fulfilled the serious adverse event definition.

[0383] Adverse Events of Special Interest

[0384] Cytokine release syndrome, neurotoxicity (including CAR-T cell-related neurotoxicity [e.g., ICANS] and other neurotoxicities), and second primary malignancy of any grade were followed as part of standard safety monitoring activities, regardless of severity or causality. These events were reported in a timely manner, irrespective of seriousness (e.g., serious and nonserious adverse events). Serious CRS or ICANS were also reported to the FDA in a timely manner regardless of causality.

[0385] Neurotoxicities and second primary malignancies were reported for the duration of the study, irrespective of treatment emergent status, and subsequently were collected in a longterm follow-up study yearly until 15 years post dosing of cilta-cel. All second primary malignancies were also reported to the FDA.

[0386] The following events were reported using the Serious Adverse Event Form within 24 hours of awareness of the event, irrespective of seriousness or causality (e.g., serious and nonserious adverse events): Grade 3 CRS; Grade 3 neurotoxicity; any grade movement and neurocognitive toxicity (i.e., parkinsonism); and any grade second primary malignancies.

[0387] Adverse events of special interest that were considered to be non-serious by the investigator were included on the serious adverse event form and in the eCRF. Grade 1 or 2 adverse events of special interest would not qualify for expedited reporting unless they meet serious adverse event criteria. All adverse events of special interest of any grade were followed until recovery or until there was no further improvement.

[0388] Delayed Adverse Events

[0389] The follow delayed AEs were collected from the time of cilta-cel administration and for the duration of study regardless of seriousness or causality, and subsequently will be collected yearly in a long-term follow-up study for up to 15 years post-infusion of cilta-cel: new malignancies and recurrence of pre-existing malignancy (all grades, regardless of seriousness or causality), with the exception of recurrent multiple myeloma (i.e., disease progression); in the event of malignancy, a tumor sample should be collected, and lentiviral integration site analysis may be performed for possible insertional mutagenesis; new incidenceor exacerbation of a pre-existing neurologic disorder (all grades, regardless of seriousness or causality); new incidence or exacerbation of a pre-existing rheumatologic or other autoimmune disorder (all grades, regardless of seriousness or causality); new incidence of Grade >3 hematologic disorder, including hypogammaglobulinemia of IgG <400 mg / dL, or any IgG level requiring replacement therapy or associated with recurrent infections (regardless of seriousness or causality); new incidence of Grade >3 infection (regardless of seriousness or causality); and all serious adverse events, regardless of causality.

[0390] All adverse events (with the exception of neurological adverse events, second primary malignancies, and HBV reactivation) and special reporting situations, whether serious or non-serious, were reported from the time a signed and dated ICF is obtained until 100 days after last administration of any study treatment or until the start of subsequent systemic anticancer therapy, if earlier, and may include contact for follow-up of safety. After 100 days, only adverse events that were considered related to study drug needed to be reported until the end of the study. Events of HBV reactivations and new neurological adverse events or exacerbation of existing neurologic adverse events were reported during the first year postdosing of cilta-cel.

[0391] All events that meet the definition of a serious adverse event will be reported as serious adverse events, regardless of whether they are protocol-specific assessments.

[0392] Expected progression of disease was not considered an adverse event (or serious adverse event). However, if determined by the investigator to be more likely related to the study treatment than the underlying disease, the clinical signs or symptoms of progression and the possibility that the study treatment is enhancing disease progression, it was reported.

[0393] All deaths not related to disease progression, regardless of attribution, were reported to the following expedited reporting procedures.

[0394] In the event of an unanticipated problem or life-threatening complications, treating investigators immediately notified the investigator. Investigators reported any adverse event (AE) that occurred after the initial dose of study treatment, during treatment, or within 100 days of the last dose of treatment on the local institutional AE form. All adverse events (with the exception of delayed AEs and HBV reactivation) and special reporting situations were reported by the subject (or, when appropriate, by a caregiver, surrogate, or the subject's legally acceptable representative) to the investigator from the time a signed and dated informed consent is obtained until 100 days after infusion of cilta-cel, regardless if PD occurs prior to Day 100, or subsequent anti -myeloma therapy was started prior to Day 100, and 100 days afterinfusion of cilta-cel. Beyond the adverse event reporting period, only SAEs regardless of causality and non-serious AEs that were considered related to a study drug needed to be reported until the end of the study except as defined for delayed AEs. In addition, events of HBV reactivations were reported during the first-year post-infusion of cilta-cel.

[0395] Adverse events were followed by the investigator, graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE Version 5.0), with the exception of CRS and ICANS. CRS was evaluated according to the American Society for Blood and Bone Marrow Transplantation (ASBMT) consensus grading. ICANS was graded using the ASBMT (American Society for Transplantation and Cellular Therapy [ASTCT]) consensus grading. In addition to capturing ICANS and CRS adverse events (graded by ASTCT consensus grading), all individual symptoms of CRS (e.g., fever, hypotension) and CAR-T cell-related neurotoxicity (e.g., depressed level of consciousness, seizures) was captured as individual adverse events and graded by CTCAE criteria. Neurotoxicity that was not temporally associated with CRS, or any other neurologic adverse events that did not qualify as ICANS, was graded by CTCAE criteria. Events of neurotoxicity or exacerbation of existing neurologic adverse events was reported for duration of the study post infusion of cilta-cel.

[0396] Changes in handwriting ( / . e. , micrographia, dysgraphia, or agraphia) was graded and reported as an adverse event in the eCRF. Should a subject experience a serious CAR-T associated neurotoxicity (either ICANS or other neurotoxicity), then a copy of the handwriting assessment log was submitted with the serious adverse event report.

[0397] Subjects with Grade 3 or higher toxicity or unresolved adverse events that led to treatment discontinuation continued to be assessed until recovery to Grade <1 or baseline, the event was deemed irreversible, the end of the study, or a maximum of 6 months, whichever came first.

[0398] Safety was measured by adverse events, laboratory test results, vital sign measurements, physical examination findings (including neurologic examination), assessment of ICE-Tool scores, handwriting assessments, assessments of cardiac function, and assessment of ECOG performance status grade. Clinically relevant changes occurring during the study were recorded on the adverse event section of the eCRF. Any clinically significant abnormalities persisting at the end of the study / early withdrawal were followed by the investigator until resolution or until a clinically stable condition was reached. Safety monitoring assessments was performed more frequently, if clinically indicated.

[0399] The study included the following evaluations of safety and tolerability according to the time points provided in the Schedule of Events. See Table 7.

[0400] Testing

[0401] Blood samples for serum chemistry and hematology were collected as shown in the Schedule of Events. See Tables 7 and 9. Disease-related laboratory evaluations are detailed in the Schedule of Events as well. The investigator reviewed the laboratory results, documented this review, and recorded any_clinically relevant changes occurring during the study in the adverse event section of the CRF. Grade 3 or higher laboratory abnormalities continued to be assessed until recovery to Grade <1 or baseline, the event was deemed irreversible, the end of study, or a maximum of 6 months, whichever came first. Laboratory certificates or accreditation and normal ranges of the laboratory facility at the site were submitted to the Sponsor Investigator before the enrollment of any subject at the site. If the subject had the laboratory assessments conducted at a laboratory facility other than the one associated with the investigational site, the investigator submitted to the Sponsor Investigator laboratory certificates or accreditation and normal ranges for that facility as well. The laboratory reports were filed with the source documents.

[0402] The following tests were performed by the local laboratory except for the calcium and albumin-adjusted calcium, which were performed at the central laboratory table:Table 16: Tests PerformedHematology _• Hemoglobin• White blood cell count• Absolute neutrophil count• Absolute lymphocyte count• Platelet count• Absolute number and % CD4• Absolute number and % CD8» CD4 / CD8 ratiog_Coagulation _Prothrombin time / international normalized ratioFibrinogenActivated partial thromboplastin timeD- dimer _Chemistry _• Sodium• Potassium• Lactic acid dehydrogenase• Blood urea nitrogen or Urea _• Creatine• AST• ALT• Gamma-glutamyl transpeptidase• Ferritin• eGFR• Triglycerides• Uric acid• Calcium and albumin-adjusted calciumb• Phosphate• Albumin• Total protein• Magnesium• Creatine phosphokinase (CPK)• C-reactive protein• Thyroid Function testingdTest• Serum (<5 lU / mL) B-hCGTests at• Serology:o Hepatitis B: HBsAg, anti-HBc, anti-HBs, HBV DNA quantification (for subjects who are anti-HBs positive without a history of vaccination or for subjects who are anti-HBc positive with or without anti-HBs positive) o Hepatitis C: Hepatitis C virus [HCV] infection is defined as:■ Positive anti-HCV antibody or■ Detectable HCV-RNA (for subjects who are anti-HCV positive) or ■ History of HCVNOTE: Participants with positive anti-HCV antibody due to prior resolved disease can be enrolled only if a confirmatory HCV-RNA test is undetectable. For participants with history of HCV infection, confirmation of sustained virologic response is required for study eligibility, defined as undetectable HCV-RNA >= 24 weeks after completion of antiviral therapy• HIVTest atHIV, Hepatitis B, Hepatitis C, HTLV, and other infectious diseases as applicable per localAbbreviations: ALT=alanine aminotransferase; AST=aspartate aminotransferase; hCG= human chorionic gonadotropin; HBsAg=hepatitis B surface antigen; anti-HBc=anti-hepatitis B core antibody, anti-HBs=anti- hepatitis B surface antibody; HBV=hepatitis B virus; HCV=hepatitis C vims, HIV=human immunodeficiency virus; HTLV=human T-cell lymphotropic vims.aDirect bilimbin if Gilbert’s disease.bPerformed by central laboratory.dThyroid assessment: TSH only, if TSH is <LLN or >ULN need free T3 and free T4 testing8CD4 / CD8 panel will be done for newly enrolled subjects and is optional as locally available.hTriglycerides is at baseline and it would be repeated as clinically indicated thereafter

[0403] 12-lead ECGs were performed as specified in the Schedule of Events. See Table 7. ECGs were obtained prior to any other study procedures planned for the same day. Additional cardiovascular assessments were performed as clinically appropriate to ensure subject safety. The investigator reviewed the results, including ECG morphology, for immediate management. Abnormalities noted at screening were included in the medical history. Assessment of cardiac function is required at screening using either echocardiogram or MUGA scan (results obtained <8 weeks before apheresis. At a minimum, included assessment of left ventricular ejection fraction (LVEF) reported as a percentage. This value was recorded in the eCRF. Temperature, pulse / heart rate, respiratory rate, blood pressure and oxygen saturation monitoring was performed as specified in the Schedule of Events. See Table 7.

[0404] The screening physical examination included, at a minimum, subject’s height, general appearance, examination of the skin, ears, nose, throat, lungs, heart, abdomen, extremities, musculoskeletal system, nervous system, and lymphatic system. Thereafter, a symptom-directed physical examination was conducted as clinically indicated at subsequent timepoints. Abnormalities were recorded in the appropriate section of the eCRF. Body weight was measured prior to infusion of cilta-cel. See Table 7. Clinically significant post-baseline abnormalities were recorded as adverse events.

[0405] The ECOG performance status scale was used to grade changes in the subject’s daily living activities and was assessed as noted in the Schedule of Events. See Table 7.

[0406] Magnetic resonance imaging (MRI) at screening or neurology consultation was considered if pre-existing disease was suspected. For subjects with prior pertinent neurologic disease (e.g., stroke, encephalitis) baseline MRI of brain and an EEG was considered. At the first sign of neurotoxicity, neurology consultation and evaluation was considered. ICANS was graded using ASBMT (ASTCT) consensus grading. Other neurologic adverse events not associated with ICANs were graded based on CTCAE version 5.0. Findings from neurologic testing that supported CAR-T cell-related neurotoxicity (eg, ICANS) were reported in the CRF. Submission of neuroimaging scans could also be requested for review.

[0407] The ICE test was developed to provide objectivity for the grading of multiple overlapping encephalopathy terms currently included on the approved CAR-T products. The ICE tool was collected as noted in the Schedule of Events (Table 7) to guide management throughout both phases of the study. It was also used to grade the severity of ICANS. All ICE scores were reported in the eCRF.

[0408] Handwriting assessment criterion have been developed to assess subjects for occurrence of the following types of changes in handwriting: micrographia, dysgraphia, or agraphia, as potential early indicators for neurotoxicity. Handwriting assessments were collected on a writing log according to Table 7. Subjects unable to write at baseline were excused from this assessment during study. The Sponsor Investigator / treating study physician was immediately notified when changes in handwriting were detected. This prompted discussion about additional assessments to further evaluate for other neurotoxicity symptoms, further workup, as well as the potential initiation of interventions. All cases of handwriting abnormalities ( / . e. , micrographia, dysgraphia, or agraphia) were reported as an adverse event in the eCRF. Should a subject experience a serious CAR-T associated neurotoxicity (either ICANS or other neurotoxicity), then a copy of the handwriting assessment log was submitted with the serious adverse event report. For visits post Day 100 that were completed remotely via Telemedicine, the handwriting assessment was administered by the mobile study personnel.

[0409] Data Reporting, Biomarker Collection, and Measurement of Effect

[0410] Correlative Studies Background

[0411] The following studies were performed: Whole-genome sequencing (WGS) of tumor cells in the bone marrow and circulating tumor cells (CTCs) at screening and at progression; single cell RNA sequencing of immune cells in the bone marrow and peripheral blood at screening, time of best response, and at 6 months, 12 months and progression; cfDNA at screening and every 3 months for the first year and then every 6 months for 5 years; ELISA for soluble BCMA; Cytometry by time of flight (CyTOF) for immune cells at screening, at best response and at time of progression; flow cytometry for the presence of the CART cells in the peripheral blood weekly for the first month, and on Day 56 and 100; and quantitative mass spectrometry at screening, 1 months, 3 months, 6 and 12 months as well as serially every 3-6 months for 5 years.

[0412] Whole genome sequencing and RNA sequencing studies were performed on tumor cells obtained at the time of screening as well as from subsequent bone marrow samples to examine clonal heterogeneity, resistant clones at best response and at time of end of study or at disease progression.

[0413] The tumor research samples were collected at the time of scheduled bone marrow biopsies. From these samples, high quality DNA and RNA for both exome sequencing and RNA sequencing of tumor cells were obtained. In brief, bone marrow (BM) aspirates were obtained after informed consent. The tumor cells were collected using CD 138+ bead selection(over 90% purity based on prior publications). For samples that had a small fraction of plasma cells, flow sorting for CD138 / CD38 / CD56 and CD 19 was used to obtain a pure malignant plasma cell population based on prior published markers of malignant plasma cells.

[0414] WGS was performed on all samples pre- and post-treatment on the clinical trial to study clonal evolution of malignant cells. This also helped address the “MGUS like” state in some cases post-therapy. Samples were multiplexed and sequenced on Illumina Novaseq to obtain an average depth of coverage of 60x for tumors and 3 Ox for germlines to have enough sensitivity for mutation detection.

[0415] All bioinformatics and statistical analyses were performed with guidance of the biostatistics and bioinformatics Core B. Briefly, BAM files aligned to the hgl9 human genome were produced using Illumina sequencing reads and the Picard pipeline. SNVs were determined using the MuTect2 algorithm, in single mode for targeted sequencing with additional filters for mutation call such as 1000 genome and COSMIC mutations. Indels and translocations were determined by the algorithms IndelLocator and dRanger, respectively. Focal as well as armlevel copy number variations were determined based on WES and subsequent application of the GISTIC algorithm. MutSigCV was used to detect candidate cancer genes using three signals of positive selection: (i) increased mutation burden as compared to a background model; (ii) clustering of mutations along the gene; and (iii) enrichment of mutations at likely functional sites. The output of MutSigCV consists of a list of the most significantly genomic events across samples. False-discovery rates (q values) < 0.1 were considered as significantly mutated. All candidate SNVs / indels / CNVs were reviewed in IGV.

[0416] Single-cell RNA sequencing (RNA-seq) of the tumor and immune microenvironment can define genotypic and phenotypic states of tumor cells and surrounding microenvironment, and that the microenvironment affected the gene expression program of tumor cells and their resistance to therapy. 10X genomics were applied in this Example 3 to evaluate the tumor microenvironment and to assess specific changes in cell type and transcriptional signature of BM niche cells that correlate with tumor progression or resistance to therapy.

[0417] It was hypothesized that (i) immune markers at baseline are predictive of PFS following immunotherapy, (ii) immunotherapy can reverse alterations of the SMM bone marrow microenvironment and (iii) immunotherapy can normalize the immune system (state of immune MRD). Single cell Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) was performed on longitudinal samples from the PRISM clinical trialproposed in Aim 1. Immune markers of progression were evaluated and the role of spatial organization in progression were assessed. It is understood that the placement of immune cells within the structure of the tumor microenvironment can affect their functionality. For example, proximity of T cells to the tumor may be required for a bispecific antibody to mediate its cytotoxic task. Here, mass cytometry (CyTOF) was used on viably frozen cells and CyTOF imaging on bone marrow slides to validate the immune markers discovered by CITE-seq and assess the role of immune cell spatial organization in response and resistance to immunotherapy. The Fluidigm Hyperion is an imaging mass cytometer that can identify numerous ligands simultaneously. CyTOF measures the abundance of metal isotopes tagged to antibodies, and amplification is achieved by a polymeric metal-chelating reagent or metal nanoparticles. This technique uses a high-resolution laser ablation system and a low-dispersion laser ablation chamber to image at a subcellular resolution of 1 pm.

[0418] CITE-sequencing refers to the parallel characterization of RNA-level and surface proteome-level expression at single-cell resolution. Specifically, the TotalSeq C feature barcoding technology was used for surface protein detection, as well as the single-cell V(D)J and 5' gene expression technology for reconstruction of TCR / BCR clonotypes and RNA expression profiling. Following mononuclear cell isolation with ficoll, CD45+ cells was magnetically separated (MACS) and frozen for batch testing. The cells were thawed, and the Chromium Controller (lOx GenomicsTM) was used for parallel sample partitioning and molecular barcoding. Cells were loaded on one channel of a Single Cell 5' Chip together with the Single Cell 5' Gel Beads (lOx GenomicsTM). Generated libraries were pooled together according to Illumina specifications and paired-end sequenced on a HiSeq 2500 platform with standard Illumina sequencing. Following data pre-processing with Cellranger, immune cell populations were identified by their RNA and surface protein expression profiles using Seurat. Using these annotations, each sample was quantified for (i) the fraction of NK cells, (ii) the ratio of memory: effector cytotoxic T cells, (iii) the abundance of regulatory T cells, (iv) the RNA and surface protein expression levels of MHC II on CD 14+ Monocytes, and (v) the degree in which novel immune alterations discovered in this larger size cohort were present. The additional immune alterations discovered using the Signature Analyzer-GPU method that uses a Bayesian version of non-negative matrix factorization to cluster scRNA-seq data and detect cell types and states and expression modules that are differentially expressed among them. The predictive value of each biomarker was quantified for the likelihood of patient progression to identify immune markers of progression.

[0419] It is understood that the placement of immune cells within the structure of the tumor microenvironment can affect their functionality. For example, proximity of T cells to the tumor may be required for a bispecific antibody to mediate its cytotoxic task. Mass cytometry (CyTOF) was used on viably frozen cells, and CyTOF imaging on bone marrow slides in order to (i) validate the immune markers discovered by CITE-seq and (ii) assess the role of immune cell spatial organization in response and resistance to immunotherapy.

[0420] A Hyperion imaging system was used to validate this signature for spatial localization and temporal changes during disease progression of specific subsets of cells. For CyTOF imaging, CyTOF panels were used on serial samples of formalin-fixed, paraffm-embed (FFPE) tissue bone marrow specimens. This multiplex imaging allows for the investigation of a larger cohort of samples and their serial bone marrow samples from the MGUS stage to SMM and overt MM. The Fluidigm Hyperion is an imaging mass spectrometer that can identify numerous ligands simultaneously. CyTOF measures the abundance of metal isotopes tagged to antibodies, and amplification is achieved by a polymeric metal-chelating reagent or metal nanoparticles. This technique uses a high-resolution laser ablation system and a low-dispersion laser ablation chamber to image at a cellular resolution of 1 pm. 40 antibodies can be included, focusing on myeloid cell populations, T cells, and NK cells. This multiplex imaging allows for the investigation of a larger cohort of samples and their serial bone marrow samples from SMM to MM. By using a panel of target protein markers specific to the populations and protein expression of interest, findings could be rapidly validated using an independent technology and cohort.

[0421] Circulating cell-free DNA (cfDNA) and tumor cells (CTCs) are considered emerging and promising approaches to capture the genomic landscape and heterogeneity of the tumor cells in bone marrow, but from blood samples. cfDNA sequencing can be challenging because of the small fragment size of DNA in the peripheral blood (average of 166bp), the low yield of DNA and the usual low allelic fraction of tumor-derived DNA among the cfDNA. Therefore, two different approaches to sequence cfDNA were developed. The first approach applies WES. After high-speed centrifugation of frozen samples to eliminate residual cells from plasma, cfDNA was extracted using the Qiagen circulating nucleic acid kit. As little as 5ng of cfDNA was then subjected to library preparation using the Kapa HyperPlus kit, which enables the preparation of libraries from small DNA fragments and minimal DNA yield. CfDNA libraries were initially qualified for further sequencing using ultra-low-pass whole genome sequencing (ULP-WGS). The ULP-WGS is a low -cost approach developed to nominate samplescontaining sufficient tumor fraction in cfDNA samples for WES. Large numbers of cfDNA libraries were multiplexed and sequenced to an average of 0.1X genome-wide sequencing coverage.

[0422] cfDNA and CTCs were subjected to library preparation using the Kapa HyperPlus kit and initially qualified for WES by ULP-WGS. The ULP-WGS was used to nominate samples containing sufficient fraction of tumor-derived DNA for WES. Qualified matched samples were hybridized to the Agilent XT v5 enrichment kit, with additional baits on MY C, IGH, IGL and IGK loci. For both cfDNA and CTCs, the coverage goal was increased to 200x, which enables the accurately call mutations even with low allelic fraction samples.

[0423] WES analyses was performed through the FireCloud, including MuTect43, IndelLocator37, dRanger, GISTIC2.044 and ABSOLUTE18 algorithms to evaluate SNVs, Indels, translocations, CNVs and mutated cancer fractions. The correlation of mutated cancer fractions between matched cfDNA, CTCs and BM tumor cells for each patient to characterize the mutational spectrum in the 3 compartments was further studied; thus, identifying their overlapping landscape as well as their potential specificities. It was expected to identify in cfDNA and CTCs more than 80% of the somatic mutations present in BM.

[0424] The level of BCMA was measured in the circulation using commercial ELISA kits at baseline (screening), and at 1 -month post-CART. The level of soluble BCMA at screening was expected to correlate with response to therapy.

[0425] To determine persistence of CAR in the peripheral blood, samples were obtained at Day 1, 3, 7, 10, 12, 14, 21, 28, 42, 42, 56, 78, 100, etc., or until undetectable.

[0426] Refer to Table 9 for more information regarding sample collection timepoints.

[0427] Samples were obtained on all participants at the time points specified in the sample collection calendar. See Tables 7 and 9. It was anticipated that over 90% of samples collected would be adequate for sequencing studies proposed based on degree of typical plasmacytosis in participants with high-risk smoldering myeloma.

[0428] Bone marrow samples were collected at the time points below specified for ID / MRD analysis: Baseline (a previous bone marrow biopsy / aspirate sample could be used for clonality sequence identification if performed within 6 months of registration. Otherwise, the clonality specimen weas performed on the screening bone marrow biopsy; if a clonality sequence was not identified through a historical sample, the participant could re-test for clonality using fresh [screening] marrow aspirate.); Day 56, Day 100; at any time that a participant underwent a premobilization bone marrow biopsy / aspirate after 6 months of treatment; after 6 months oftreatment; after 1 year of infusion; after 18 months of infusion; after 24 months of infusion; completion of Study; and for subjects with suspected CR or at the time of CR, and then yearly for subjects that remain on study up to disease progression.

[0429] See Table 17 for a summary of correlative studies from the bone marrow biopsy (20 cc research sample).

[0430] The clonoSEQ Assay is an in vitro diagnostic assay that utilizes NGS to identify frequency and distribution of clonal sequences consistent with a malignant lymphocyte population in a sample.

[0431] Minimal Residual Disease (MRD) refers to the measurable number of cancer cells that remain in a person during and following treatment. Clinical practice guidelines in select hematological malignancies recognize that MRD status is a reliable indicator of clinical outcome and response to therapy. Studies in select hematological malignancies have demonstrated the strong correlation between MRD and risks for relapse, as well as the prognostic significance of MRD measurements during and after therapy. In multiple myeloma, MRD assessment after each treatment stage is recommended (e.g., after induction, high-dose therapy / ASCT, consolidation, maintenance). MRD tests may also be initiated at the time of suspected complete response. The assay has been validated with the Illumina NextSeq 500 and 550 and is FDA approved for use in MM. The clonoSEQ Assay is a next-generation sequencing (NGS) based assay that identifies rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCLl / IgH (J) and BCL2 / IgH (J) sequences. The assay also includes primers that amplify specific genomic regions present as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content. Testing begins with gDNA extracted from the specimen supplied. Extracted gDNA quality is assessed and rearranged immune receptors are amplified using a multiplex PCR. Reaction-specific index barcode sequences for sample identification are added to the amplified receptor sequences by PCR. Sequencing libraries are prepared from barcoded amplified DNA, which are then sequenced by synthesis using NGS. Raw sequence data are uploaded from the sequencing instrument to the Adaptive analysis pipeline. These sequence data are analyzed in a multi-step process: first, a sample’s sequence data are identified using the sample index sequences. Next, data are processed using a proprietary algorithm with in-line controls to remove amplification bias. When the clonoSEQ Clonality (ID) assessment is conducted, the immune repertoire of the sample is checked for the presence of DNA sequences specific to “dominant” clone(s) consistent with the presence of a lymphoid malignancy. Each sequence that is being consideredfor MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value that, together with its abundance relative to other sequences, is used to assign the sequence to a sensitivity bin which will be used in the estimation of the reported LoD and LoQ on the patient report. During clonoSEQ Tracking (MRD) assessment, the complete immunoglobulin receptor repertoire is again assessed, and the previously identified dominant clonotype sequence(s) are detected and quantified to determine the sample MRD level. The clonoSEQ Assay MRD assessment measures residual disease in a biologic sample. Following completion of these data processing steps, a report is issued. A Clonality (ID) report indicates the presence of dominant sequences residing within a presumed malignant lymphocyte clonal population, as identified in the baseline (diagnostic or high disease burden) sample from a patient. After one or more dominant sequence(s) have been identified in a baseline sample, subsequent samples from the same patient can be assessed for MRD after which a Tracking (MRD) report is generated. The MRD is expressed as a frequency that quantifies the level of residual disease based on the number of remaining copies of the initially dominant sequence(s) relative to the total number of nucleated cells in the sample.

[0432] Mass spectrometry is a novel and a more sensitive method to detect presence of M-protein in serum, allowing for extended M-protein quantification at very low levels. The MiRAMM (monoclonal Ig rapid accurate mass measurements) has been proven to be an effective method of detection of low-level M protfluein, demonstrating continued decreased intensity of miRAMM leads to longer PFS in the post-transplant setting. The present Exampled assessed mass spectrometry using Binding Site assay for disease response assessment and correlate with MRD status as a secondary end point.

[0433] Biomarker Studies

[0434] Biomarker assessments focused on several objectives: 1) evaluate infused CAR-T cell subsets and activation markers including, but not limited to, CD4+, CD8+, CD25+, central memory, effector memory cells; 2) determine the ability of cilta-cel to induce MRD negativity in subjects with smoldering myeloma who have achieved CR; 3) serum or plasma proteomic profiling of cytokines (such as IL-6, IL- 15, and IL- 10) and other immune related proteins (such as perforin and granzymes); 4) whole genome sequencing and single cell sequencing and immune single cells sequencing along with immunophenotyping of biomarkers of response / resistance on myeloma cells (such as BCMA and PD-L1); 5) determine the clinical benefit (ORR, DOR, TTR, PFS, and OS) of cilta-cel in subjects with cytogenetic modifications(dell7p, t(4; 14), t( 14; 16), or other high-risk molecular subtypes); and 6) immunophenotyping of immune cells subsets such as CD4+ and CD8+ T-cells, regulatory T-cells, B and NK-cells.

[0435] Additional biomarker samples could be collected to help understand an unexplained adverse event including but not limited to serum or peripheral blood mononuclear cells (PBMCs) from whole blood. Additional sample(s) for cytokines were collected as clinically indicated.

[0436] The potential presence of RCL was evaluated from whole blood samples of subjects treated with cilta-cel. To monitor if RCL emerges from cilta-cel, whole blood from participants were evaluated using a quantitative PCR assay against the LV vesicular stomatitis virus-G gene at approximately 3 months, 6 months, and 12 months after cilta-cel infusion (as specified in the Schedule of Activities). Yearly review of medical history was generally sufficient for the participant for up to 15 years after cilta-cel infusion in the present study (until end of study) or in a separate long-term follow-up study. If any post-infusion samples were positive, further RCL analysis and more extensive participant follow-up was undertaken. Additional samples were collected triggered by events which may be relevant, but not limited, to RCL per clinical assessment as specified in the Schedule of Activities. See Tables 7 and 9. In addition, a blood sample was collected for RCL evaluation at SPM onset.

[0437] Investigators could request additional material from previously collected bone marrow samples during or after study completion for a retrospective analysis. For subjects diagnosed with an SPM, a tumor sample was collected, and DNA, RNA, or protein analysis would be performed to investigate the presence of LV elements.

[0438] Additional samples (including but not limited to blood, tissue, tumor, etc.) could be requested as clinically indicated. Additionally, investigators would receive a sample of plasmacytoma if a plasmacytoma biopsy was performed for any reason, including during screening. Subjects who had a lumbar puncture as part of their neurologic work up would have cerebral spinal fluid for additional testing by investigators. In this case, such analyses was specific to research related to the study treatment(s) or diseases being investigated. If a subject died and an autopsy was performed, specimens were requested by investigators for analysis, as allowed by local regulations.

[0439] Biomarker samples were collected as indicated in Table 9.

[0440] Pharmacodvnamic / Predictive Markers

[0441] Baseline characteristics of the cilta-cel T cell subsets and dynamic changes / persistence and activation of cilta-cel positive viable T-cells may be associated withthe depth and durability of response. An evaluation of these cell populations could be performed by flow cytometry or cytometry by time of flight (CyTOF) or both and correlated with response. Additional immunophenotyping could be performed on bone marrow aspirate and whole blood samples to evaluate expression of biomarkers on myeloma cells (such as BCMA and PD-L1) and immune cell populations (such as CD4+ and CD8+ T-cells) by flow cytometry, or CyTOF, or next generation sequencing (whole exome and RNA sequencing) or both. T-cell receptor (TCR) sequencing could be performed to study T-cell clonality that may affect drug response. Samples could be characterized by gene expression profding and somatic mutation analysis by next generation sequencing (whole exome and RNA sequencing) to evaluate potential biomarkers that may correlate with response. Samples could be evaluated by other similar technologies to evaluate protein or RNA expression or for somatic DNA analysis.

[0442] Circulating serum biomarkers present following chemotherapy conditioning and following infusion of CAR-T cells have been associated with response to some CAR-T cellbased therapies. Evaluation of cytokines (such as IL-6, IL-15, IL-10, and IFN- y) and other circulating proteins (such as granzymes and perforin) were performed to identify potential biomarkers of hemophagocytic lymphohistiocytosis (HLH) and / or indicative of cytokine release syndrome (CRS) including, triglycerides, interleukin (IL)-6, C-reactive protein, ferritin and / or fibrinogen, IL- 10, interferon (IFN)-gamma.

[0443] Minimal Residual Disease

[0444] 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 will be used to define the myeloma clones, and post-treatment samples were used to evaluate MRD negativity. A fresh bone marrow aspirate was collected at screening as the baseline sample. If the NGS MRD method was unavailable, or determined to be scientifically inferior, then alternative methods for MRD assessment were utilized. Additional timepoints for MRD assessment are reflected in the Schedule of Events (Table 9).

[0445] In the event fresh bone marrow aspirate was not collected at baseline, or if the fresh aspirate does not yield a usable clone, non-decalcified diagnostic tissue (bone marrow aspirate slides or formalin-fixed paraffin embedded tissue) should be collected for calibration of myeloma cells to facilitate the assessment of the MRD endpoints by NGS.

[0446] Pharmacokinetics (PK) and Immunogenicity

[0447] The goal of the pharmacokinetic assessment of cilta-cel in this study was to evaluate pharmacokinetic parameters, and immunogenicity effects on the pharmacokinetic profiles and parameter values. Pharmacokinetic / pharmacodynamics, dose-response (safety and efficacy) relationships were explored. Immunogenicity assessments were also be utilized in these evaluations. See Schedule of Events (Table 9) for sample collection instructions and schedule.

[0448] Blood and serum samples were collected for cilta-cel pharmacokinetics, and immunogenicity (antibodies to cilta-cel) assessment as specified in the Schedule of Events (Table 9). Also, pharmacokinetic and immunogenicity samples were collected at the time onset of suspected CRS or CAR-T cell-related neurotoxicity (e.g., ICANS) regardless of causality (See Table 9, Footnote g). In addition, pharmacokinetic and immunogenicity samples were collected following the end of study treatment as shown in Table 9. The exact dates and times of blood sampling were recorded on the laboratory requisition form.

[0449] Venous blood samples were collected for measurement of CAR-T positive cellular concentration of cilta-cel.

[0450] Bone marrow samples were collected for measurement of cellular concentrations of cilta-cel (see Schedule of Events, Tables 7 and 9).

[0451] Post-dose blood and bone marrow samples were analyzed to determine CAR-T positive cellular concentration of cilta-cel using specific and sensitive assay methods that were validated by or under the supervision of investigators.

[0452] The detection and characterization of antibodies to cilta-cel were performed using a validated assay method by or under the supervision of investigators. Other analyses could be performed to characterize immunogenicity.

[0453] Blood and bone marrow samples were collected for the measurement of cilta-cel cellular concentrations for pharmacokinetic analyses (Schedule of Events, Table 9) Pharmacokinetic parameters were estimated for individuals, and descriptive statistics were calculated. Correlation of Cmax and AUC with dose could also be explored. Pharmacokinetic parameters included, but were not limited to, AUCinf, AUC(O-t), AUCtau, Cmax, half-life, and Tmax parameters were calculated if sufficient data was available for estimation.

[0454] Antibodies to cilta-cel was evaluated in serum samples collected during the Treatment Phase (Schedule of Events, Table 9). Additionally, serum samples were also collected at the final visit from subjects who discontinued treatment or were withdrawn from the study.

[0455] Serum samples were screened for antibodies binding to cilta-cel and the titer of confirmed positive samples were reported. Other analyses were performed to verify the stability of antibodies to cilta-cel or further characterize the immunogenicity of cilta-cel.

[0456] PK and anti-drug antibody (ADA) samples were collected at the following timepoints. Flow Cytometry PK Blood: Pre-apheresis, Within 7 days prior to first dose of conditioning regimen, Pre-cilta-cel infusion, Day 3, Day 7, Day 10, Day 12, Day 14, Day 21, Day 28, Day 42, Day 56, Day 78, Day 100, Month 6, Month 9, Year 1 post-cilta-cel, and At time of progressive disease (if applicable) or at End of Study ,_ADA Blood: Pre-cilta-cel infusion, Day 14, Day 28, Day 56, Day 78, Day 100, Month 6, and at time of progressive disease (if applicable) or at End of Study.

[0457] Measurement of Effect

[0458] In this study, symptomatic MM that requires therapy is defined as one of the following criteria (CRAB* and Myeloma defining events< MDE): increased calcium levels (corrected serum calcium >0.25 mmol / dL above the upper limit of normal or >.275 mmol / dL); renal insufficiency (attributable to myeloma); anemia (Hgb 2g / dL below the lower limit of normal or <10g / dL); bone lesions (lytic lesions or generalized osteoporosis with compression fractures) or any MYELOMA DEFINING EVENTS (MDE) as follows: clonal bone marrow plasma cell percentage*> 60%; an abnormal FLC-ratio >100 (involved kappa) or <0.01 (involved lambda); 2 or more focal lesions on MRI or PET-CT studies. Participants with CRAB criteria that are attributable to conditions other than the disease under study will not be considered development of symptomatic MM.

[0459] In this study, participants must have measurable disease. The disease response was assessed using criteria based on the International Working Group Uniform Response Criteria in this section. If the only measurable parameter is serum immunoglobulins free light chain (FLC), the participant was followed by FreeLiteTM Disease Response Criteria provided in this section.

[0460] The same method of assessment and technique was used for disease measurement at baseline and during follow-up. Disease response was confirmed by two consecutive assessments.

[0461] Measurable disease is disease that can be measured either by serum or urinary evaluation of the monoclonal component or by serum assay of FLC and is defined by at least one of the following three measurements: serum M-protein > 0.5 g / dl; urine M-protein > 200mg / 24 h; serum FLC assay: Involved FLC level > 10 mg / dl (> 100 mg / 1) provided serum FLC ratio is abnormal.

[0462] If a patient had measurable disease, but not by IMWG standards listed above, the patient could be evaluable after discussion with the Sponsor Investigator.

[0463] All baseline evaluations were performed on the first day of initial therapy. Response was assessed by M-protein quantification, protein electrophoresis and immunofixation from serum and a 24-hour urine collection. A serum sample for FreeLiteTM testing was obtained. In addition, bone marrow aspiration and biopsy, as well as skeletal survey was performed to determine overall response or confirm response.

[0464] The same method of assessment and technique was used for disease measurement at baseline and during follow-up.

[0465] A confirmation measurement for disease response assessments was required in this protocol.

[0466] Response criteria for all categories and subcategories of response except CR were applicable only to participants who have ‘measurable’ disease. All response categories required two consecutive assessments made at any time before the institution of any new therapy; all categories also required no known evidence of progressive or new bone lesions if radiographic studies were performed. Radiographic studies were not required to satisfy these response requirements.

[0467] Disease Response Criteria

[0468] See Table 17 for criteria for response to MM treatment.

[0469] Forthose participants being followed by serum free light chain (and NO measurable serum or urine M-protein), which were immunofixation negative at enrollment, normalization of serum free light chain ratio. Normalization is defined as the serum free light chain ratio being within the normal range. If the serum free light chain ratio was not within the normal range, but the individual kappa and lambda light chain values were within normal range, this was considered CR.

[0470] If only measurable parameter was serum immunoglobulins free light chain (FLC), EITHER of the following changes qualify as partial response: a 50% decrease in the difference between involved and uninvolved FLC levels; or a 50% decrease in the level of involved FLC AND a 50% decrease (or normalization) in the ratio of involved / uninvolved FLC.

[0471] If only measurable parameter is serum immunoglobulins free light (FLC), either of the following qualify as progression: 50% increase in the difference between involved anduninvolved FLC levels from the lowest response level, which must also be an absolute increase of at least 10 mg / dL; or 50% increase in the level of involved FLC AND a 50% increase in the ratio of involved / uninvolved FLC from the lowest response level.

[0472] MRD was measured in all participants at the following timepoints: baseline (A previous bone marrow biopsy / aspirate sample may be used for clonality sequence identification if performed within 6 months of registration. Otherwise, the clonality specimen were performed on the screening bone marrow biopsy); Day 28 and Day 100; at any time a participant undergoes a pre-mobilization bone marrow biopsy / aspirate; after 6 months of treatment; after 1 year of treatment; after 18 months of treatment; after 24 months of treatment; completion of Study; 1 Year Follow Up (1 year after treatment ends); and 2 Years Follow Up and 3 Year Follow Up.

[0473] MRD was carried out according to the clonoSEQ method.

[0474] For MM diagnostic samples, genomic DNA was amplified using locus-specific primer sets for the immunoglobulin heavy-chain locus (IGH) complete (IGH-VDJH), IGH incomplete (IGH-DJH), and immunoglobulin K locus (IGK). The amplified product was subjected to sequencing, and the sequences and frequencies of the different clonotypes in the sample was obtained. Myeloma gene rearrangements were identified. Participants in whom a high-frequency myeloma clone (>5%) was not identified were excluded from the MRD analysis. MRD was assessed in participants with a high-frequency myeloma clone using the IGH-VDJH and IGK or IGH-VDJH, IGH-DJH, and IGK assays. Once the absolute amount of total cancer-derived molecules present in a sample was determined, a final MRD measurement was calculated, providing the number of cancer-derived molecules per 1 million cell equivalents. In cases in which 2 or more tumor clones existed, the clone with the highest MRD value was reported. Molecular CR was defined according to the International Myeloma Working Group (IMWG) consensus recommendations.

[0475] A negative result is defined as the absence of clonal plasma cells by NGS on bone marrow aspirate in which presence of a clone is defined as less than 2 identical sequencing reads obtained after DNA sequencing of bone marrow aspirates using the LymphoSIGHT platform with a minimum sensitivity of 1 in 105 nucleated cells or higher.

[0476] Mass spectrometry is a novel and a more sensitive method to detect presence of M-protein in serum, allowing for extended M-protein quantification at very low levels. Investigators assessed for presence of M protein by Mass spectrometry for participants at selected visits, at EOT, suspected CR, and during follow up as feasible. This also included atthat time of their MRD analysis (at 6 months, 1 year and at 2 years and beyond if funding and time permits). Investigators aimed to see if mass spectrometry negativity correlates with MRD negativity in this patient population. The study assessed mass spectrometry using The Binding Site assay (MALDI-TOF).

[0477] A negative result is defined as the absence of detectable monoclonal protein on MALDI-TOF.

[0478] CLIA approved monoclonal gammopathy by quantitative immunopurification mass spectrometry (QIP-MS) were conducted on the blood at timepoints selected above as well as at the times where MRD testing was performed via Adaptive ClonoSeq. This assay undergoes extensive validation according to Clinical and Laboratory Standards Institute guidelines before they are judged suitable for routine clinical use.

[0479] The samples that were identified to be negative or equivocal for an M-spike during their CLIA approved mass spectrometry analysis were shipped and LC-MS was performed for confirmation of the absence of a monoclonal protein according to the DTA / MTA executed agreements as part of the correlative analysis plan.

[0480] Results from time of achieving CR was correlated with next generation sequencing for minimal residual disease testing as part of secondary aims of this study. Samples were exhausted upon LCMS, and data returned to the study team in the form of secure data files.

[0481] MRD negative CR is defined by undetectable clonality sequence by next generation sequencing using clonoSEQ assay

[0482] Duration of Response and Endpoints

[0483] The duration of overall response is measured as the time from initiation of first response to first documentation of disease progression or death whichever occurs first. Participants who had not progressed or died are censored at the date last known progression-free.

[0484] The duration of overall CR is progression or death from initial CR. Participants who had not progressed or died are censored at the date last known progression-free.

[0485] Time to progression is defined as the time of infusion until progression. Participants who had died without evidence of progression are censored in the TTP analysis at the time of death and participants who are alive without progression were censored at the last disease assessment.

[0486] Overall survival (OS) is defined as the time from CART therapy to death. Alive participants were censored at the date last known alive.

[0487] Progression-Free Survival (PFS) is defined as the time from CART therapy to the disease progression or death from any cause, whichever occurs first. Participants who had not progressed or died are censored at the date last known progression-free.

[0488] Central review of disease response assessments was not planned outside of routine and targeted monitoring efforts.

[0489] Statistical Considerations

[0490] This was a feasibility study to evaluate the safety and efficacy of cilta-cel in the treatment of smoldering multiple myeloma. This study enrolled up to 2 safety run-in cohorts, each cohort enrolling 3 participants per the decision plan described. The study plans to enroll additional participants based on the decision plan stated below for a total accrual goal of 20 participants.

[0491] 3 participants were enrolled each in two safety run-in phases and observe them for dose-limiting toxicities for 60 days. Treatment of participants within each safety run-in cohort was staggered by 6 weeks.

[0492] Following the safety run-in, additional participants will be enrolled until a total of 20 are treated to evaluate efficacy. This portion of the study will utilize a single-stage, singlearm binomial endpoint design with all participants treated at the recommended dose. All participants regardless of dose level will be assessed for safety and toxicity endpoints.

[0493] The analysis populations for this study are defined as follows.

[0494] All Treated Analysis Set: This set consists of subjects who received cilta-cel infusion and will be considered as the primary analysis set for safety summaries.

[0495] Pharmacokinetic Analysis Set: This set consists of all subjects who received cilta-cel infusion and have at least 1 post-dose pharmacokinetic sample.

[0496] Immunogenicity Analysis Set: This set consists of all subjects who received cilta-cel infusion and have at least 1 post-dose immunogenicity sample.

[0497] Sample Size Determination

[0498] The efficacy outcome will be performed on all 20 participants receiving treatment.

[0499] The study will use a single-stage, exact binomial design. The complete response rate (CR) of at least 40% will be considered promising while an CR of 5% or less will be considered non-promising. Twenty eligible participants will be treated. If three or fewer CRs are observed, the regimen will be considered non-promising, and the study will be unsuccessful. If at least four CRs are observed in 20 participants, the study will be considered successful. This design has an overall power and one-sided type-I error of > 0.89 and 0.016, respectively. The primaryoutcome of CR will be reported as a proportion with 90 and 99% exact binomial confidence intervals. With a total sample size of 20 participants, the single-stage exact 99% confidence interval for CR will be no wider than 56%.

[0500] Overall response rate (ORR) is defined as the proportion of subjects who achieve a PR or better according to the IMWG criteria.

[0501] VGPR or better response rate (sCR+CR+VGPR) is defined as the proportion of subjects who achieve a VGPR or better response according to the IMWG criteria.

[0502] MRD negative disease (MRD-) is defined as zero residual sequence cells detected at a level of one million cells. At time of final analysis, the 2-year MRD- rate will be summarized as a proportion with a 90% exact binomial confidence interval.

[0503] Mass spectrometry (MS) is an alternative method of MRD assessment that has shown comparable results to NGS MRD in multiple myeloma. This study requires testing by both methods and have which will allow for more stringent definition of MRD negativity as a combined end point by adding a more sensitive method of assessing MRD.

[0504] Duration of response (DOR) will be calculated among responders (with a PR or better response) from the date of initial documentation of a response (PR or better) to the date of first documented evidence of progressive disease, as defined in the IMWG criteria. Relapse from CRby positive immunofixation or trace amount of M-protein is not considered as disease progression. Disease evaluations will continue beyond relapse from CR until disease progression is confirmed. For subjects who have not progressed, data will be censored at the last disease evaluation before the start of any subsequent anti-myeloma therapy.

[0505] Time to response (TTR) is defined as the time between date of the initial infusion of cilta-cel and the first efficacy evaluation that the subject has met all criteria for PR or better. For subjects without response, data will be censored either at the date of progressive disease, or in the absence of progressive disease, at the last disease evaluation before the start of subsequent anti-myeloma therapy.

[0506] Progression-free survival (PFS) defined as the time from the date of the initial infusion of cilta-cel to the date of first documented disease progression, as defined in the IMWG criteria, or death due to any cause, whichever occurs first. For subjects who have not progressed and are alive, data will be censored at the last disease evaluation before the start of any subsequent anti -myeloma therapy.

[0507] Overall survival (OS) is measured from the date of the initial infusion of cilta-cel to the date of the subject’s death. If the subject is alive or the vital status is unknown, then the subject’s data will be censored at the date the subject was last known to be alive.

[0508] Time-to-event endpoints were assessed using the Kaplan-Meier method, and estimates were reported with 95% confidence intervals. Continuous variables were summarized using the number of observations, mean, standard deviation, coefficient of variation, median, and range as appropriate. Categorical values were summarized using the number of observations and percentages as appropriate.

[0509] Subgroup Analysis

[0510] CR rate will be summarized for the following subgroups of participants: age 65 years or older; high risk cytogenetics based on t(4: 14), t( 14: 16), 17p deletion or p53 mutation, +lq amplification (3 groups: high risk, low risk, or Fish failure)

[0511] Safety Analysis

[0512] All safety analyses were performed on data from the all-treated analysis set. The baseline value for safety assessment is defined as the value collected at the time closest to, but prior to, the start of cilta-cel infusion. The safety parameters evaluated were the incidence, severity, and type of adverse events, clinically significant changes in the subject’s physical examination findings, vital signs measurements, and clinical laboratory results. Exposure to investigational product and reasons for discontinuation of study treatment were tabulated. Adverse events were summarized by system organ class, preferred term, worst grade experienced by the subject, and by dose level.

[0513] The verbatim terms used in the eCRF by investigators to identify adverse events were coded using the Medical Dictionary for Regulatory Activities (MedDRA). Treatment-emergent adverse events are adverse events with onset after cilta-cel infusion or that are a consequence of a pre-existing condition that has worsened since baseline (Time of study consent). All reported treatment-emergent adverse events were included in the analysis. For each adverse event, the percentage of subjects who experienced at least 1 occurrence of the given event were summarized. Summaries, listings, datasets, or subject narratives could be provided, as appropriate, forthose subjects who died, experience an adverse event of special interest, discontinued treatment due to an adverse event, or who experienced a severe or a serious adverse event.

[0514] Adverse events that occur after administration of the conditioning regimen and before cilta-cel infusion were summarized and listed separately.

[0515] Laboratory data was summarized by type of laboratory test. Reference ranges and markedly abnormal results (specified in the Statistical Analysis Plan or Data Presentation Plan) were used in the summary of laboratory data. Descriptive statistics were calculated for each laboratory analyte at baseline and for observed values and changes from baseline at each scheduled time point. Worst toxicity grade during treatment was presented according to NCI-CTCAE Version 5.0. Change from baseline to the worst toxicity grade experienced by the subject during the study were provided as shift tables. A listing of subjects with any laboratory results outside the reference ranges were provided.

[0516] The interpretation of the ECGs as determined by a qualified physician (investigator or qualified designee) were summarized at scheduled time points.

[0517] Descriptive statistics of temperature, pulse / heart rate, respiratory rate, and blood pressure (systolic and diastolic) values and changes from baseline were summarized at each scheduled time point. The percentage of subjects with values beyond clinically important limits were summarized.

[0518] Pharmacokinetic / Pharmacodvnamic and Immunogenicity Analyses

[0519] All concentrations below the lowest quantifiable concentration or missing data were labeled as such in the concentration database. Concentrations below the lowest quantifiable concentration were treated as zero in the summary statistics. Descriptive statistics were used to summarize CAR-T positive cell count at each sampling timepoint.

[0520] If sufficient data are available, population-PK analysis of peripheral cilta-cel cellular concentration could be performed. If the population-PK analysis was conducted, details were given in a population-PK analysis plan and the results of the analysis were presented in a separate report. Exposure-response analyses could also be performed.

[0521] If sufficient data was available, then other PK / pharmacodynamic modeling could be performed, including exploring the relationship between cilta-cel cellular concentrations, pharmacodynamic markers (e.g., sBCMA, M-protein) and endpoints of clinical efficacy and safety.

[0522] The incidence of anti -cilta-cel antibodies was summarized for all subjects who receive cilta-cel and have appropriate samples for detection of antibodies to cilta-cel (i.e., subjects with at least 1 sample obtained after the infusion of cilta-cel). The results were summarized by dose level for subjects with appropriate samples for the detection of antibodies to cilta-cel.

[0523] Immunogenicity analyses is descriptive in nature and includes the number and percentage of subjects who developed anti-cilta-cel antibodies. The effect of anti-cilta-cel antibodies on pharmacokinetics, safety, and efficacy could also be evaluated.

[0524] Patient-Reported Outcome Assessments

[0525] The EORTC QLQ-C30, EORTC QLQ-MY20 (4 items), EQ-5D-5L utility and visual analog scores, PGIC, and PGIS were descriptively summarized at each time point. Meaningful and sustained improvement in subject’s HRQoL comparative to their baseline health status were evaluated using established meaningful change thresholds. Within-group change of the PRO endpoints were assessed by change from baseline (screening phase) using mixed models for repeated measures.

[0526] Sample Size. Accrual Rate, and Study Duration

[0527] A total of 20 eligible evaluable participants over the safety run-in and expansion portions will be enrolled and assessed for primary and secondary endpoints. It is expected that approximately 18 months of active accrual with approximately 2 participants per month would be needed; participants will be followed for PFS and survival after the primary endpoint of CR rate had been assessed.

[0528] Table 19 provides an estimated breakdown of participants from different race, sex, and ethnicity to consist of this study accrual goal.

[0529] The final analyses of CR endpoints (estimating the 2-year CR rate and estimating the response distribution) will be conducted at the end of the study which occurred after either all participants have completed the 2-year assessment for response or if all participants have been removed from follow-up for response, whichever occurrs first.Example 4: Early Safety and Efficacy of CAR-T cell Therapy in Precursor Myeloma: Results using Ciltacabtagene Autoleucel in High-Risk Smoldering Myeloma

[0530] Introduction

[0531] CAR-T cell therapy has been used in relapsed multiple myeloma (MM) with deep and durable responses but some patients still eventually relapse. Investigators hypothesized that the use of Ciltacabtagene Autoleucel (cilta-cel), a BCMA-directed CAR T-cell therapy, could be safe and highly effective in patients with high-risk smoldering myeloma (HR-SMM), where the tumor burden is lower with less genomic complexity and the immune system is more fit, potentially leading to less toxicity and improved durable remissions in this high risk precursor condition.

[0532] Methods

[0533] This is a phase II, single arm study of cilta-cel in HR-SMM, defined per the Mayo / IMWG 20-2-20 model, IMWG risk score of 9 or greater, high-risk FISH, evolving pattern or the PETHEMA criteria. Patients with >40% bone marrow plasmacytosis were excluded. Patients received lymphodepletion (LD) with fludarabine and cyclophosphamide followed by cilta-cel infusion. The first 6 patients comprise a safety run-in cohort, with the first 3 patients dosed at a lower target dose of 0.5 x 106CAR-positive T-cells / kg. If there were no dose limiting toxicities (DLTs), the next 3 patients were dosed at the standard target dose of 0.75 x 106CAR-positive T-cells / kg. The primary objective of this study was to determine the safety of cilta-cel in HR-SMM. Secondary objectives assessed efficacy, MRD negativity rates and progression-free survival. Correlative studies assessed in vivo proliferation, persistence, and activation of CAR T-cells after infusion as well as characteristics of malignant cells and the tumor microenvironment.

[0534] Results

[0535] Six patients were treated in the safety run-in with a median follow up of 6 months (60 days to 1 year). The median age was 55 years (range 53-66) with 3 females and 3 males. The isotypes were IgG (4), light chain only (1) and IgD (1). No DLTs were observed in the safety run-in for both 0.5 and 0.75 x 106CAR-positive T-cells / kg doses.

[0536] Grade 3 or greater hematologic toxicities were expected, transient and attributed to LD (neutropenia 100%, anemia 33%, thrombocytopenia 17%). Grade 3 or greater non-hematologic toxicities included AST and ALT increased (17%, during CRS and expected), hypertriglyceridemia (17%), and lymphocytosis (17%). No grade 3 or greater infections were observed.

[0537] All patients experienced low grade cytokine release syndrome (CRS) (Grade 1 in 67% and grade 2 in 33%) and no high grade CRS events were observed. Tocilizumab was administered to 4 patients and 2 patients received dexamethasone. There were no instances of ICANS, Parkinsonism or secondary malignancies to date. One patient experienced grade 1 Bell’s palsy that was self-limiting and resolved within 2 weeks. One patient experienced grade 4 immune related thrombocytopenia secondary to fludarabine, which resolved within 2 weeks after treatment with dexamethasone, IVIG and romiplostim.

[0538] CAR T-cells expanded in all patients with peak expansion on days 12-14 after infusion (median 15 days, FIG. 8A). By flow cytometry, median absolute CAR+ T cell count at peak expansion was 3.8 K / uL (range 0.7 - 30 K / ul). As expected, most of the T cells wereCAR+ T cells at peak expansion (mean 83%) (FIG. 8B). CAR T cells were predominately CD4+ with an effector memory phenotype and remained detectable at 3 months in 2 patients. CAR T cell expansion and persistence were similar at both dose levels. A plot of CD4+ and CD8+ CAR T cell expansion over 1 year is shown in FIG. 8C, with detectable levels of CAR T cells at about 6 months in multiple patients and at about 1 year in at least one patient.

[0539] All patients achieved MRD negativity at 10'6by day 28 and MRD negativity was sustained in all patients by a median follow up of 6 months (60 days to 1 year) without any evidence of progression. The overall response rate is 100%, with complete response rate of 50% and responses deepening over time, consistent with delayed paraprotein clearance in the setting of MRD negativity. By a median follow up of 10.5 months, the complete response rate was 100%. Stem cell collection was successful in all eligible patients with an average stem cell yield of 8.94xl06CD34+ cells / kg.

[0540] FIG. 9 shows a plot of each patient’s response to therapy. Overall, response continued to deepen over time, and to date all patients have achieved a strident complete response (sCR). MRD negativity occurs early, prior to achieving the best serological response. The first three patients (Pl, P2, P3 in FIG. 9) have sustained MRD negativity after 1 year of follow up, with the final three patients (P4, P5, P6) being MRD negative at the time of their last follow up. No patient has developed biochemical or SLIM-CRAB progression to date.

[0541] To highlight the effectiveness of treatment, one of the six patients is reviewed in more detail. The patient is a 54-year-old active male with kappa light chain smoldering multiple myeloma (SMM). The patient was diagnosed with SMM in 2023 and placed on observation but had a continued rise in kappa light chains and an increased in bone marrow plasmacytosis in 2024. The patient had FISH with monosomy 13 and t(l 1 ; 14) and was considered high risk per 20-2-20 criteria with an evolving pattern. FIG. 10 shows the levels of kappa light chains in this patient before and after treatment with cilta-cel. After cilta-cel infusion, the detectable levels of kappa light chains are near or at zero.

[0542] ConclusionsThis is the first study of CAR T-cell therapy in a precursor cancer setting, where cilta-cel was used as a primary therapy with no induction therapy for patients with HR-SMM. There were no DLTs in the safety run-in cohort. CRS was minimal, no ICANS were observed, and there were no grade 3 or greater infections. All patients achieved MRD negative (10‘6) disease.Example 5: Spatially Resolved Tumor-Immune Interactions Predict Immunotherapy Outcomes in Smoldering and Relapsed Myeloma

[0543] Introduction

[0544] T cell-redirecting therapies, including chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs), have transformed the treatment landscape of relapsed / refractory multiple myeloma (RRMM) and are being explored in earlier disease stages, including high-risk smoldering multiple myeloma (SMM). Emerging evidence suggests that the spatial organization of tissues critically shapes outcomes to these immunotherapies. In this study, we used spatial proteomics on bone marrow (BM) core biopsies to (1) map the BM microenvironment across myeloma stages, (2) assess how baseline spatial features influence immunotherapy (BsAb or CAR-T) response, and (3) characterize longitudinal remodeling of the BM niche during treatment.

[0545] Methods

[0546] 121 BM trephine biopsies from 47 high-risk SMM patients treated with teclistamab or cilta-cel, and 19 RRMM patients treated with BsAbs (14 teclistamab, 2 elranatamab, 3 talquetamab) were analyzed. Thirty-six patients had at least one matched post-treatment biopsy. A total of 196 regions of interest (ROIs) were profiled using a 32-marker IMC panel (Hyperion XTi, Standard BioTools). Cell segmentation was performed with cellpose-sam and batch-corrected using ComBat. Marker-based cell type annotation identified 27 distinct phenotypes across 1,116,993 single cells. Cellular neighborhoods, defined as the 10 pm microenvironment around each cell, were inferred via Mini Batch K-means clustering. Spatial co-localization was evaluated using squidpy permutation testing.

[0547] Results

[0548] SMM and RRMM cohorts exhibited expected differences: all RRMM patients had prior BCMA-directed therapy, 42% had extramedullary disease, and the median number of prior treatment lines was five (range 2-13). In contrast, SMM patients were mostly treatment-naive and without end-organ damage. High-risk cytogenetics were similarly distributed (17.5% vs 26.7%, p=0.7).

[0549] As an initial quality control step, plasma cell (PC) infiltration detected by IMC was validated against independent pathologist...

Claims

What is claimed is:

1. A method of treating smoldering multiple myeloma in a subject in need thereof, the method comprising administering ciltacabtagene autoleucel to the subject.

2. The method of claim 1 , wherein the smoldering multiple myeloma is a high-risk smoldering multiple myeloma.

3. The method of claims 1 or 2, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.1 to about 1.0 x 106CAR-positive viable T cells / kg.

4. The method of claim 3, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.75 x 106CAR- positive viable T cells / kg.

5. The method of claim 3, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.5 x 106CAR-positive viable T cells / kg.

6. The method of claim 3, wherein ciltacabtagene autoleucel is administered to the subject at a dose of about 0.3 x 106CAR-positive viable T cells / kg.

7. The method of any one of claims 1 to 6, wherein ciltacabtagene autoleucel is administered in a single infusion.

8. The method of any one of claims 1 to 7, wherein the maximum total dose of ciltacabtagene autoleucel is 1 x 108CAR-positive viable T-cells.

9. The method of any one of claims 1 to 8, wherein the subject achieves minimum residual disease (MRD) negative status by about 28 days after administration of ciltacabtagene autoleucel.

10. The method of claim 9, wherein the subject sustains minimum residual disease (MRD) negative status after about 6 months after administration of ciltacabtagene autoleucel.

11. The method of claim 9, wherein the subject sustains minimum residual disease (MRD) negative status after about 1 year after administration of ciltacabtagene autoleucel.

12. The method of any one of claims 1 to 8, wherein the method further comprises treating the subject for an adverse event after administering ciltacabtagene autoleucel, wherein the adverse event comprises a nonhematologic adverse event, a hematologic adverse event, a treatment-emergent adverse event, or any combination thereof.

13. The method of claim 12, wherein the adverse event comprises neutropenia, thrombocytopenia, anemia, lymphopenia, hypertriglyceridemia, lymphocytosis, an upper respiratory tract infection, nasopharyngitis, sinusitis, rhinitis, tonsillitis, pharyngitis, laryngitis, pharyngotonsillitis, COVID-19, COVID-19 pneumonia, asymptomatic COVID-19, neutropenic sepsis, progressive multifocal leukoencephalpathy, septic shock, respiratory failure, pulmonary embolism, a lower respiratory tract / lung infection, pneumonia, bronchitis, nausea, hypogammaglobulinemia, diarrhea, fatigue, headache, constipation, hypokalemia, asthenia, peripheral edema, decreased appetite, peripheral sensory neuropathy, back pain, arthralgia, pyrexia, dyspnea, insomnia, or any combination thereof.

14. The method of claim 12, wherein the nonhematologic adverse event comprises an infection and / or a nonhematologic adverse event other than an infection.

15. The method of claim 12, wherein the treatment- emergent adverse event comprises cytokine release syndrome (CRS).

16. The method of claim 15, wherein the maximum toxicity grade of the CRS is Grade 1 or Grade 2.

17. The method of claim 15 or 16, wherein treatment of the CRS comprises intravenous fluids, tocilizumab, methylprednisolone, dexamethasone, oxygen, a corticosteroid, a vasopressor, or any combination thereof.

18. The method of any one of claims 1-17, wherein CAR-T cells in the blood of the subject peak at about 12 days to about 14 days after administering the ciltacabtagene autoleucel to the subject.

19. The method of claim 18, wherein CAR-T cells in the blood of the subject peak at about 14 days after administering the ciltacabtagene autoleucel to the subject.

20. The method of any one of claims 1-19, wherein CAR-T cells in the blood of the subject peak at a median absolute CAR+T cell count at peak expansion of about 0.7 K / uL to about 30 K / uL after administering the ciltacabtagene autoleucel to the subject.

21. The method of claim 20, wherein CAR-T cells in the blood of the subject peak at a median absolute CAR+T cell count at peak expansion of about 3.8 K / uL after administering the ciltacabtagene autoleucel to the subject.

22. The method of any one of claims 1-21, wherein CAR-T cells in the blood of the subject remain detectable about 3 months after administering the ciltacabtagene autoleucel to the subject.

23. The method of any one of claims 1-21, wherein CAR-T cells in the blood of the subject remain detectable about 6 months after administering the ciltacabtagene autoleucel to the subject.

24. The method of any one of claims 1-21, wherein CAR-T cells in the blood of the subject remain detectable about one year after administering the ciltacabtagene autoleucel to the subject.

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