Compositions and methods for use in the treatment of cancer

Abstract

Provision of chimeric antigen receptors (CARs) including centirilin. [Solution] Disclosed are a chimeric antigen receptor (CAR) containing centirinn (i.e., CARTyrin), a transposon encoding the CAR and CARTyrin of this disclosure, cells modified to express the CAR and CARTyrin of this disclosure, and methods for producing and using them for adoptive cell therapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefit of U.S. Provisional Application No. 63 / 009,569, filed on 14 April 2020. The contents of this application are incorporated herein by reference in their entirety.

[0002] This disclosure relates to molecular biology, more specifically to chimeric antigen receptors, transposons containing one or more CARs, and methods for producing and using them.

[0003] Incorporation of sequence lists The contents of a text file named "POTH-057_001WO_SequenceListing.txt", created on April 13, 2021, and measuring 54.6KB, are incorporated into this specification in their entirety by reference. [Background technology]

[0004] There has long been a pressing but unmet need in the art for a method of guiding the specificity of immune cells without using traditional antibody sequences or fragments thereof. This disclosure provides an excellent chimeric antigen receptor. [Overview of the Initiative]

[0005] This disclosure provides a method for treating cancer, comprising administering to a subject a first composition comprising a population of T cells expressing a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-recognition domain that specifically binds to a B cell maturation antigen (BCMA), and a second composition comprising an anti-CD20 agent. In some embodiments, the method further comprises a third composition comprising at least one lymphocyte-depleting agent. In some embodiments, the anti-CD20 agent is rituximab, ofatumumab, ocrelizumab, iodine i131 tositumomab, obinutuzumab, or ibritumomab. In preferred embodiments, the anti-CD20 agent is rituximab. In some embodiments, the antigen-recognition domain comprises centintin, scFv, a single-domain antibody, VH, or VHH. In some embodiments, the antigen-recognition domain comprises centintin. In some embodiments, the antigen-recognition domain comprises VH.

[0006] This disclosure also provides a method for treating cancer, comprising administering to a subject a first composition comprising a population of T cells expressing a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-recognition domain, and a second composition comprising an anti-CD20 agent. In some embodiments, the first composition comprises a population of T cells expressing two or more CARs. In some embodiments, each CAR in the two or more CARs binds to a different antigen. In some embodiments, the method further comprises a third composition comprising at least one lymphocyte-depleting agent. In some embodiments, the anti-CD20 agent is rituximab, ofatumumab, ocrelizumab, iodine i131 tositumomab, obinutuzumab, or ibritumomab. In preferred embodiments, the anti-CD20 agent is rituximab. In some embodiments, the antigen-recognition domain comprises centinrin, scFv, a single-domain antibody, VH, or VHH. In some embodiments, centinrin specifically binds to B cell maturation antigen (BCMA). In some embodiments, VH specifically binds to BCMA. In some embodiments, centinlin specifically binds to prostate-specific membrane antigen (PSMA). In some embodiments, scFv binds to mucin 1 (MUC-1). In some embodiments, scFv binds to MUC1-C.

[0007] In some embodiments, the method provides at least a 50% reduction in the anti-drug antibody (ADA) response to the first composition in patients compared to patients who are administered the first composition but not the second composition.

[0008] In some embodiments, the method provides at least a 75% increase in the persistence of the first composition in patients compared to patients administered the first composition but not the second composition. In some embodiments, the method provides at least a 90% increase in the persistence of the first composition in patients compared to patients administered the first composition but not the second composition. In some embodiments, persistence is measured by the area under the plasma concentration curve (AUC).

[0009] In some embodiments, the first composition is administered as multiple infusions, each infusion comprising a total dose divided into a first infusion and a second infusion, i) the first infusion comprising about one-third of the total dose, and ii) the second infusion comprising about two-thirds of the total dose, administered at least 10 days after the first infusion.

[0010] In some embodiments, the first composition is administered as multiple infusions, each infusion comprising a total dose divided into a first infusion and a second infusion, i) the first infusion comprising about two-thirds of the total dose, and ii) the second infusion comprising about one-third of the total dose, administered at least 10 days after the first infusion.

[0011] In some embodiments, the first composition is administered as multiple infusions, each infusion comprising a total dose divided into a first infusion, a second infusion, and a third infusion, i) the first infusion comprising about one-third of the total dose, ii) the second infusion comprising about one-third of the total dose and administered at least 10 days after the first infusion, and iii) the third infusion comprising about one-third of the total dose and administered at least 10 days after the second infusion.

[0012] In some embodiments, the time between the first injection and the second injection, or between the second injection and the third injection, is at least one, two, three, four, five, or six weeks.

[0013] In some embodiments, the first composition, the second composition, and the third composition are administered sequentially. In some embodiments, the first composition, the second composition, and / or the third composition are administered simultaneously. In some embodiments, the third composition is administered before the first composition.

[0014] In some embodiments, the third composition is administered in two or more doses. In some embodiments, the third composition is administered once daily, with the first dose of the third composition administered at least five days before the first infusion of the first composition. In some embodiments, the third composition is administered three, four, and five days before the first infusion of the first composition.

[0015] In some embodiments, the second composition is administered before the first composition. In some embodiments, the second composition is administered in two or more doses. In some embodiments, the first dose of the second composition is administered 12 days before the first infusion of the first composition, the second dose of the second composition is administered 5 days before the first infusion of the first composition, and subsequent doses are administered once a week for at least 8 weeks after the first infusion of the first composition.

[0016] In some embodiments, the subject has not been previously treated with an anticancer drug. In some embodiments, the subject has not received an anticancer drug within i) two weeks prior to administration of the first dose of the second composition, or ii) within five half-lives of the anticancer drug prior to administration of the first dose of the second composition.

[0017] In some embodiments, the subject does not receive an anticancer drug after the administration of the first infusion of the first composition. In some embodiments, the subject has not been previously treated with an immunosuppressant. In some embodiments, the subject has not received an immunosuppressant i) within two weeks prior to the administration of the first dose of the second composition, or ii) within five half-lives of an immunosuppressant prior to the administration of the first dose of the second composition. In some embodiments, the subject does not receive an immunosuppressant after the administration of the first infusion of the first composition.

[0018] In some embodiments, the subject has not been previously treated with an anti-inflammatory agent. In some embodiments, the subject does not receive an anti-inflammatory agent within i) two weeks prior to administration of the first dose of the second composition, ii) one week prior to administration of the first dose of the second composition, or ii) five half-lives of an immunosuppressant prior to administration of the first dose of the second composition. In some embodiments, the subject does not receive an anti-inflammatory agent after administration of the first infusion of the first composition. In some embodiments, the anti-inflammatory agent is a corticosteroid. In some embodiments, the corticosteroid is prednisone, which is administered systemically at a dose of at least 5 mg / day.

[0019] In some embodiments, the subject has not been previously treated with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the subject does not receive G-CSF or GM-CSF within i) two weeks prior to administration of the first dose of the second composition, or ii) within five half-lives of G-CSF or GM-CSF prior to administration of the first dose of the second composition. In some embodiments, the subject does not receive G-CSF or GM-CSF after the last infusion of the first composition and / or within two months after the last infusion of the first composition.

[0020] In some embodiments, the subject has not been previously treated with herbal medicine. In some embodiments, the subject has not received herbal medicine within two weeks prior to the administration of the first dose of the second composition. In some embodiments, the subject has not received herbal medicine after the administration of the last infusion of the first composition and / or within two months after the administration of the last infusion of the first composition.

[0021] In some embodiments, the first lymphocyte depleting agent of the third composition and the second lymphocyte depleting agent of the third composition are administered simultaneously. In some embodiments, the first lymphocyte depleting agent of the third composition and the second lymphocyte depleting agent of the third composition are administered sequentially. In some embodiments, the first lymphocyte depleting agent and the second lymphocyte depleting agent are administered on the same day, the first lymphocyte depleting agent is administered intravenously over a period of 30 minutes, and the second lymphocyte depleting agent is administered intravenously over a period of 30 minutes.

[0022] In some embodiments, the first lymphocyte depleting agent or the second lymphocyte depleting agent is cyclophosphamide or fludarabine. In some embodiments, the dose of the third composition is i) 100 mg / m 6 , 6 , 6 , 200 mg / m 2 , 300 mg / m 2 [[ID=­10]], 400 mg / m 2 , or 500 mg / m 2 of cyclophosphamide, ii) 10 mg / m 2 , 20 mg / m 2 , 30 mg / m 2 , 40 mg / m[[ID=2­1]] 2 , or 50 mg / m 2 of fludarabine, or a combination thereof. In some embodiments, the dose of the third composition comprises 300 mg / m 2 of cyclophosphamide and 30 mg / m 2 of fludarabine.

[0023] In some embodiments, the first composition comprises at least cells 0.1×10 6 , 0.2×10 6 , 0.25×10 6 , 0.5×10 6 , 0.6×10 6 , 0.7×10 6 , 0.75×10 6 , 0.8×10 6 , 0.9×10 6 , 1×10 6 , 2×10 6 , 3×10 6 , 4×106 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 10×10 6 , 11×10 6 , 12×10 6 , 13×10 6 , 14×10 6 , 15×10 6 , 16×10 6 , 17×10 6 , 18×10 6 , 19×10 6 , or 20×10 6 The total dose is administered in units / kg of body weight of the subject. In some embodiments, the first infusion, second infusion, and / or third infusion of the first composition delivers the first composition to approximately 1 × 10 cells. 5 pieces / mL ~ approx. 5×10 7 It is administered using an infusion bag containing the first composition at a concentration of cells / mL. In some embodiments, the infusion bag contains approximately 3 × 10 cells of the first composition. 5 pieces / mL ~ approx. 2.4×10 7 Contains at a concentration of 1 / mL.

[0024] In some embodiments, the first infusion, the second infusion, and / or the third infusion are administered by intravenous infusion at a flow rate of approximately 0.5 mL / min to approximately 30 mL / min. In some embodiments, the flow rate is approximately 1 mL / min to approximately 20 mL / min. In some embodiments, the total duration of the first infusion, the second infusion, and / or the third infusion is approximately 5 minutes to approximately 30 minutes.

[0025] In some embodiments, the dose of the second composition is 100 mg / m². 2 , 125 mg / m² 2 , 150 mg / m² 2 , 175 mg / m² 2 , 200 mg / m² 2 , 225 mg / m² 2 , 275 mg / m² 2 , 300 mg / m² 2 , 325 mg / m² 2, 375 mg / m² 2 , 400 mg / m² 2 , 425 mg / m² 2 , 450 mg / m² 2 , 475 mg / m² 2 , or 500 mg / m² 2 It contains rituximab. In a preferred embodiment, the dose of the second composition is 375 mg / m². 2 It is rituximab.

[0026] In some embodiments, the second composition is administered by intravenous infusion at a flow rate of approximately 25 mg / hour to approximately 500 mg / hour. In some embodiments, the first dose of the second composition is administered by intravenous infusion at a flow rate of 50 mg / hour, with the flow rate increased every 30 minutes up to a maximum of 400 mg / hour. In some embodiments, the second dose and subsequent doses of the second composition are administered by intravenous infusion at a flow rate of approximately 100 mg / hour, with the flow rate increased every 30 minutes up to a maximum of approximately 400 mg / hour.

[0027] In some embodiments, acetaminophen, an antihistamine, and methylprednisolone are administered 30 minutes before each dose of the second composition.

[0028] In some embodiments, cancer is a blood cancer. In some embodiments, blood cancer is multiple myeloma. In some embodiments, multiple myeloma is relapsed multiple myeloma or refractory multiple myeloma.

[0029] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the solid tumor is breast cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, or kidney cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0030] This disclosure provides a unit dose infusion bag containing a 250 mL composition containing a population of T cells expressing CAR, the CAR comprising an antigen recognition domain containing centrin that specifically binds to BCMA, and the concentration of the composition is approximately 3 × 10⁶ cells. 5 cells / mL~cells approx. 2.4×10 7 The concentration is cells / mL.

[0031] This disclosure provides a unit dose infusion bag containing a 250 mL composition containing a population of T cells expressing CAR, the CAR comprising an antigen recognition domain including VH that specifically binds to BCMA, and the concentration of the composition is approximately 3 × 10⁶ cells. 5 cells / mL~cells approx. 2.4×10 7 The concentration is cells / mL.

[0032] This disclosure provides a unit dose infusion bag containing a 250 mL composition containing a population of T cells expressing CAR, the CAR comprising an antigen recognition domain containing centrin that specifically binds to PSMA, and the concentration of the composition is approximately 3 × 10⁶ cells. 5 cells / mL~cells approx. 2.4×10 7 The concentration is cells / mL.

[0033] This disclosure provides a unit dose infusion bag containing a 250 mL composition containing a population of T cells expressing CAR, the CAR comprising an antigen recognition domain including scFv that specifically binds to MUC1-C, and the concentration of the composition is approximately 3 × 10⁶ cells. 5 cells / mL~cells approx. 2.4×10 7 The concentration is cells / mL. [Brief explanation of the drawing]

[0034] [Figure 1] This is a schematic diagram showing the 7676 base pair piggyBac CARTyrin construct of the present disclosure, which includes a transposon containing CARTyrin (containing the CD8a signal peptide, centinlin, CD8a hinge sequence, and transmembrane sequence, as well as the CD3z costimulatory domain). [Figure 2] This is a schematic diagram of the amino acid sequence of the P-BCMA-101 construct of this disclosure. [Figure 3A] This is a schematic diagram of the nucleic acid sequence of the P-BCMA-101 construct of this disclosure. [Figure 3B] This is a schematic diagram of the nucleic acid sequence of the P-BCMA-101 construct of this disclosure. [Figure 4] This disclosure includes a schematic diagram showing the construction of CARTyrin, and a table comparing the characteristics of centirinn and the antibody. [Figure 5A] This is a series of cell sorting plots showing CARTyrin expression after electroporation using 5 μg of CARTyrin mRNA. [Figure 5B] The left panel shows a series of cell sorting plots illustrating CARTyrin function after addition using the control K562 cell line and the BCMA-expressing H929 cell line, while the right panel shows graphs quantifying the plots in the left panel (with additional data from addition using the BCMA-expressing U266 cell line). [Figure 5C] This graph shows CARTyrin activity as a function of the amount of mRNA used during T cell electroporation. [Figure 6] This is a schematic diagram showing the timeline of an in vivo tumor loading test using A08 CARTyrin in mice. [Figure 7] This is a set of graphs showing the complete (100%) survival rate of mice treated with A08 CARTyrin. Tumor burden was assessed by the presence of the M protein. No detectable M protein was found in protected animals. [Figure 8] This is a schematic diagram illustrating an example of the inducible cleavage caspase 9 polypeptide of the present disclosure. [Figure 9]A series of flow cytometry plots, shown 12 days post-nucleofection, show the amount of cells migrating from the region of living cells (gated lower-right quadrant) to the region aggregated by apoptotic cells (upper-left quadrant), as a function of increasing doses of the inducer (AP1903) in cells modified to express the therapeutic agent (CARTyrin) alone or in combination with the inducible caspase polypeptide of the present disclosure (encoded by an iC9 construct, also known as the "safety switch") introduced into cells by the piggyBac(PB) transposase. [Figure 10] A series of flow cytometry plots, shown 19 days post-nucleofection, show the amount of cells migrating from the region of living cells (gated lower-right quadrant) to the region aggregated by apoptotic cells (upper-left quadrant), as a function of increasing doses of the inducer (AP1903) in cells modified to express the therapeutic agent (CARTyrin) alone or in combination with the inducible caspase polypeptide of the present disclosure (encoded by an iC9 construct, also known as the "safety switch") introduced into cells by the piggyBac(PB) transposase. [Figure 11] Figure 9 (left graph) or Figure 10 (right graph) is a set of graphs that represent the quantification of the aggregated results. Specifically, these graphs show the effect of the iC9 safety switch on cell viability as a function of the concentration of the iC9 switch inducer (AP1903) for each modified cell type, on day 12 (Figure 9 and left graph) or day 19 (Figure 10 and right graph). [Figure 12A] This graph shows the stable expression and function of BCMA CARTyrin. Figure 12A is a flow cytometry plot showing CARTyrin surface expression in P-BCMA-101 after piggyBac (PB) rearrangement. The mock represents primary BCMA / Fc / biotin-free. [Figure 12B] Figure 12B is a flow cytometry plot showing increased CARTyrin expression in restimulated P-BCMA-101 T cells. [Figure 12C]Figure 12C is a graph showing in vitro cell death of BCMA+(H929) cells by P-BCMA-101 cells. [Figure 12D] Figure 12D is a flow cytometry plot showing the proliferation of P-BCMA-101 cells against BCMA-expressing cell lines. [Figure 13A] This line graph shows the in vivo tumor growth and survival rate of MM.1S-Luc tumor-bearing NSG mice treated with P-BCMA-101 in a GLP safety study. Female NSG mice were IV-transplanted with M.1SBCMA+MM cells, and 17-19 days later, they were IV-administered either 4×10⁶ P-BCMA-101 cells (low dose) (n=19) (red) or 12×10⁶ P-BCMA-101 cells (high dose) (green) or a vehicle (n=10) (black). No tumors were found (blue) (n=20). On day 29, 10 mice from each treatment group were euthanized and submitted for pathology. Figure 13A is a graph showing the mean ± mean standard error (SEM) of the bioluminescence imaging data. [Figure 13B] Figure 13B is a graph showing bioluminescence imaging data for each individual mouse from each treatment group. [Figure 13C] Figure 13C shows the survival curves illustrating the survival percentages of mice from three treatment groups. [Figure 13D] Figure 13D is a graph showing the change in body weight of mice from each treatment group. [Figure 14] This is a schematic diagram of the overall study design for a single dose of P-BCMA-101. [Figure 15] This is a schematic diagram of the clinical production and process plan for P-BCMA-101. [Figure 16] This is a schematic diagram of the overall study design for P-BCMA-101 cyclic administration in Cohorts A and C. In Phase 1 - Cycle administration, multiple doses of P-BCMA-101 are administered intravenously over two 2-week cycles. [Figure 17]This is a schematic diagram of the overall study design for P-BCMA-101 cyclic administration in Cohort B. In Phase 1 - cyclic administration, multiple doses of P-BCMA-101 are administered intravenously over three 2-week cycles. [Figure 18] This is a schematic diagram of the overall study design for Phase 1 combination therapy with P-BCMA-101. In Phase 1 combination therapy, P-BCMA-101 will be administered in combination with the approved therapies, lenalidomide and rituximab. [Figure 19A] This graph shows the time course of P-BCMA-101 copies / ugDNA in multiple myeloma patients treated with P-BCMA-101 monotherapy. [Figure 19B] This graph shows the time course of P-BCMA-101 copies / ugDNA in multiple myeloma patients treated with P-BCMA-101 in combination with rituximab. [Modes for carrying out the invention]

[0035] This disclosure provides a method for treating cancer, comprising administering to a subject a first composition comprising a population of T cells expressing a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain, and a second composition comprising an anti-CD20 agent.

[0036] In some embodiments, the antigen recognition domain of the CAR includes centintin, scFv, a single-domain antibody, VH, or VHH.

[0037] In some embodiments, the antigen-recognition domain specifically binds to B cell maturation antigen (BCMA) or tumor necrosis factor receptor superfamily member 17 (TNFRSF17). In some embodiments, the antigen-recognition domain includes sentinelin that specifically binds to BCMA. Non-limiting examples of sentinelin that specifically binds to BCMA are disclosed in PCT Publication 2018 / 014038. In some embodiments, the sentinelin that specifically binds to BCMA includes the amino acid sequence of SEQ ID NO: 41.

[0038] In some embodiments, the antigen recognition domain includes a VH that specifically binds to BCMA. Non-limiting examples of VHs that specifically bind to BCMA are disclosed in PCT Publication 2019 / 126574.

[0039] In some embodiments, the antigen recognition domain specifically binds to prostate-specific membrane antigen (PSMA). In some embodiments, the antigen recognition domain includes centintrinn that specifically binds to PSMA. Non-limiting examples of centintrinn that specifically bind to PSMA are disclosed in PCT Publication WO2019 / 173636.

[0040] In some embodiments, the antigen-recognition domain specifically binds to mucin 1 (MUC-1). Human MUC1 is a heterodimeric glycoprotein, translated as a single polypeptide, and cleaved in the endoplasmic reticulum into N- and C-terminal subunits (MUC1-N and MUC1-C). In some embodiments, the antigen-recognition domain includes an scFv that specifically binds to MUC1-C. Non-limiting examples of centintil that specifically binds to MUC1-C are disclosed in PCT application PCT / US2020 / 066121 and PCT publication 2018 / 014039.

[0041] The centiriins of this disclosure bind specifically to antigens. Using the chimeric antigen receptors of this disclosure, which include one or more centiriins that bind specifically to antigens, the specificity of cells (e.g., cytotoxic immune cells) can be directed to specific antigens.

[0042] The centinlin of this disclosure may include a consensus sequence containing LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (Sequence ID 1).

[0043] The chimeric antigen receptors of this disclosure may include signal peptides of human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR. The hinge / spacer domains of this disclosure may include hinge / spacer / stalks of human CD8α, IgG4, and / or CD4. The intracellular domains or endodomains of this disclosure may include the intracellular signaling domain of human CD3ζ and may further include human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. Exemplary transmembrane domains include, but are not limited to, human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domains.

[0044] As used herein, the term “P-BCMA-101” refers to centiririn that binds to BCMA, CARTyrin that binds to BCMA, CAR that specifically binds to BCMA, or T cells or populations of T cells that express CARTyrin or CAR that specifically binds to BCMA. In some cases, “P-BCMA-101” refers to a construct that encodes CAR or CARTyrin that binds to BCMA (including, for example, surrounding elements such as DHFR and iC9).

[0045] In some embodiments, P-BCMA-101 CARTyrin comprises a human CD8α signal peptide containing the amino acid sequence of SEQ ID NO: 3, an antigen recognition region containing centrin that specifically binds to BCMA containing the amino acid sequence of SEQ ID NO: 41, a human CD8α hinge region containing the amino acid sequence of SEQ ID NO: 10, a human CD8α transmembrane region containing the amino acid sequence of SEQ ID NO: 4, a human 4-1BB costimulatory domain containing the amino acid sequence of SEQ ID NO: 8, and a CD3 zeta costimulatory domain containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, P-BCMA-101 CARTyrin comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, P-BCMA-101 CARTyrin is encoded by a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 44.

[0046] This disclosure provides genetically modified cells, such as T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells (including G-CSF-mobilized peripheral T cells), and umbilical cord blood (UCB)-derived T cells, that are specific for one or more antigens by introducing the CAR and / or CARTyrin of this disclosure. The cells of this disclosure can be modified by electrophoretic transfer of a plasmid containing a transposon encoding the CAR or CARTyrin of this disclosure and a sequence encoding the transposase of this disclosure (preferably the sequence encoding the transposase of this disclosure is an mRNA sequence). Examples of transposons encoding CARTyrins are described in PCT / US2019 / 021224, which is incorporated herein by reference in its entirety. Examples of transposons encoding CAR are described in PCT / US2018 / 066936 and PCT / US2017 / 042457, which are incorporated herein by reference in their entirety, respectively.

[0047] The transposons of this disclosure are maintained or incorporated by episomes into the genome of recombinant / modified cells. The transposons may be part of a two-component piggyBac system that utilizes transposons and transposases for enhanced nonviral gene delivery. In certain embodiments of this method, the transposon is a plasmid DNA transposon having two cis-regulatory insulator elements adjacent to a sequence encoding a chimeric antigen receptor. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, particularly in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac® or Super piggyBac® (SPB) transposase.

[0048] In certain embodiments of the methods of the present disclosure, the transposon is a plasmid DNA transposon having two cis-regulatory insulator elements that encode sequences of an adjacent antigen receptor. In certain embodiments, the transposon is a piggyBac transposon. In certain embodiments, in particular, in embodiments where the transposon is a piggyBac transposon, the transposase is a piggyBac® or Super piggyBac® (SPB) transposase. In certain embodiments, in particular, in embodiments where the transposase is a Super piggyBac® (SPB) transposase, the sequence encoding the transposase is an mRNA sequence.

[0049] In certain embodiments of the methods disclosed herein, the transposase enzyme is piggyBac(PB) transposase enzyme. piggyBac(PB) transposase enzyme is [Table 1] It contains, or may contain, an amino acid sequence that is identical to at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between.

[0050] In certain embodiments of the method disclosed herein, the transposase enzyme has the following sequence: [Table 2] The piggyBac(PB) transposase enzyme contains or consists of an amino acid sequence having an amino acid substitution at one or more of the 30th, 165th, 282nd, or 538th positions.

[0051] In a particular embodiment, the transposase enzyme is a piggyBac(PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at two or more of the positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 12. In a particular embodiment, the transposase enzyme is a piggyBac(PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at three or more of the positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 12. In a particular embodiment, the transposase enzyme is a piggyBac(PB) transposase enzyme comprising or consisting of an amino acid sequence having an amino acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 12. In a particular embodiment, the amino acid substitution at position 30 of the sequence of SEQ ID NO: 12 is a substitution from isoleucine (I) to valine (V). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 12 is a substitution from glycine (G) to serine (S). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 12 is a substitution from methionine (M) to valine (V). In certain embodiments, the amino acid substitution at position 538 of the sequence of SEQ ID NO: 12 is a substitution from asparagine (N) to lysine (K).

[0052] In certain embodiments of the methods of the present disclosure, the transposase enzyme is Super piggyBac®(sPBo) transposase enzyme. In certain embodiments, the Super piggyBac®(sPBo) transposase enzyme of the present disclosure comprises or may comprise the amino acid sequence of Sequence ID No. 12, wherein the amino acid substitution at position 30 is an isoleucine (I) to valine (V) substitution, the amino acid substitution at position 165 is a glycine (G) to serine (S) substitution, the amino acid substitution at position 282 is a methionine (M) to valine (V) substitution, and the amino acid substitution at position 538 is an asparagine (N) to lysine (K) substitution. In certain embodiments, the Super piggyBac®(sPBo) transposase enzyme is [Table 3] It contains, or may contain, an amino acid sequence that is identical to at least 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between.

[0053] In certain embodiments of the methods of the present disclosure, including those embodiments, in which the transposase contains the above mutations at positions 30, 165, 282, and / or 538, piggyBac® or Super The piggyBac® transposase enzyme may further include amino acid substitutions at one or more of the following positions in the sequence of SEQ ID NO: 12 or SEQ ID NO: 2: 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591. In certain embodiments, including those embodiments in which the transposase includes the above mutations at positions 30, 165, 282, and / or 538, the piggyBac® or Super piggyBac® transposase enzyme may further include amino acid substitutions at one or more of the following positions: 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552, and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from serine (S) to asparagine (N). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from alanine (A) to serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from alanine (A) to threonine (T). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from isoleucine (I) to tryptophan (W). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from serine (S) to proline (P).In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from arginine (R) to proline (P). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from cysteine ​​(C) to alanine (A). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from cysteine ​​(C) to leucine (L). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from tyrosine (Y) to lysine (K). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from tyrosine (Y) to histidine (H). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from phenylalanine (F) to leucine (L). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from phenylalanine (F) to isoleucine (I). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from phenylalanine (F) to valine (V). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to leucine (L). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from alanine (A) to glycine (G). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from phenylalanine (F) to tryptophan (W). In certain embodiments, the amino acid substitution at position 207 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from valine (V) to proline (P). In certain embodiments, the amino acid substitution at position 209 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from valine (V) to phenylalanine (F). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to phenylalanine (F).In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from leucine (L) to arginine (R). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 12 or SEQ ID NO: 12 is a substitution from valine (V) to lysine (K). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from phenylalanine (F) to leucine (L). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from proline (P) to lysine (K). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from asparagine (N) to serine (S). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from leucine (L) to tryptophan (W). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from leucine (L) to tyrosine (Y). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from leucine (L) to phenylalanine (F). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to alanine (A). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to valine (V). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from proline (P) to isoleucine (I). In certain embodiments, the amino acid substitution at position 311 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution of proline (P) to valine. In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution of arginine (R) to lysine (K).In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from threonine (T) to glycine (G). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from tyrosine (Y) to arginine (R). In certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from tyrosine (Y) to valine (V). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from cysteine ​​(C) to glycine (G). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from cysteine ​​(C) to leucine (L). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from aspartic acid (D) to histidine (H). In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from valine (V) to isoleucine (I). In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to tyrosine (Y). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from leucine (L) to phenylalanine (F). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from serine (S) to lysine (K). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to leucine (L). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to isoleucine (I). In certain embodiments, the amino acid substitution at position 552 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from valine (V) to lysine (K). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from alanine (A) to threonine (T).In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from glutamine (Q) to proline (P). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from glutamine (Q) to arginine (R).

[0054] In certain embodiments of the methods of the present disclosure, including those embodiments in which the transposase includes the above mutations at positions 30, 165, 282, and / or 538, the piggyBac® transposase enzyme or Super piggyBac® transposase enzyme may further include amino acid substitutions at one or more of positions 103, 194, 372, 375, 450, 509, and 570 of the sequence of SEQ ID NO: 12 or SEQ ID NO: 2. In certain embodiments of the methods of the present disclosure, including those embodiments in which the transposase includes the above mutations at positions 30, 165, 282, and / or 538, the piggyBac® transposase enzyme or Super piggyBac® transposase enzyme may further include two, three, four, five, six, or more amino acid substitutions at positions 103, 194, 372, 375, 450, 509, and 570 of the sequence of SEQ ID NO: 12 or SEQ ID NO: 2. In certain embodiments, including those embodiments in which the transposase includes the above mutations at positions 30, 165, 282, and / or 538, the piggyBac® transposase enzyme or Super piggyBac® transposase enzyme may further include amino acid substitutions at positions 103, 194, 372, 375, 450, 509, and 570 of the sequence of SEQ ID NO: 12 or SEQ ID NO: 2. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from serine (S) to proline (P). In certain embodiments, the amino acid substitution at position 194 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from methionine (M) to valine (V). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from arginine (R) to alanine (A). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from lysine (K) to alanine (A). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution from aspartic acid (D) to asparagine (N).In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution of serine (S) to glycine (G). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 12 or SEQ ID NO: 2 is a substitution of asparagine (N) to serine (S). In certain embodiments, the piggyBac® transposase enzyme may include a substitution of methionine (M) to valine (V) at position 194 of SEQ ID NO: 12. In certain embodiments, including those embodiments in which the piggyBac® transposase enzyme may include a substitution of methionine (M) to valine (V) at position 194 of SEQ ID NO: 12, the piggyBac® transposase enzyme may further include amino acid substitutions at positions 372, 375, and 450 of the sequence of SEQ ID NO: 12 or SEQ ID NO: 2. In certain embodiments, the piggyBac® transposase enzyme may include a substitution of methionine (M) to valine (V) at position 194 of SEQ ID NO: 12, a substitution of arginine (R) to alanine (A) at position 372 of SEQ ID NO: 12, and a substitution of lysine (K) to alanine (A) at position 375 of SEQ ID NO: 12. In certain embodiments, the piggyBac® transposase enzyme may include a substitution of methionine (M) to valine (V) at position 194 of SEQ ID NO: 12, a substitution of arginine (R) to alanine (A) at position 372 of SEQ ID NO: 12, a substitution of lysine (K) to alanine (A) at position 375 of SEQ ID NO: 12, and a substitution of aspartic acid (D) to asparagine (N) at position 450 of SEQ ID NO: 12. Scaffold protein.

[0055] The protein scaffolds of this disclosure may originate from fibronectin type III (FN3) repeat proteins, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods for producing and using them. In a preferred embodiment, the protein scaffold consists of a consensus sequence of multiple FN3 domains from human tenascin C (hereinafter "tenascin"). In a more preferred embodiment, the protein scaffold of the present invention is a consensus sequence of 15 FN3 domains. The protein scaffolds of this disclosure can be designed to bind to a variety of molecules, for example, cell target proteins. In a preferred embodiment, the protein scaffolds of this disclosure can be designed to bind to wild-type and / or mutant epitopes of an antigen.

[0056] The protein scaffolds of this disclosure may include additional molecules or portions, such as the Fc region of an antibody, an albumin-binding domain, or other portions that affect the half-life. In further embodiments, the protein scaffolds of this disclosure may be bound to nucleic acid molecules capable of encoding a protein scaffold.

[0057] This disclosure provides at least one method for expressing at least one protein scaffold based on a consensus sequence of multiple FN3 domains in host cells, comprising culturing the host cells described herein under conditions in which at least one protein scaffold is expressed in a detectable and / or recoverable amount.

[0058] This disclosure provides at least one composition comprising (a) a protein scaffold and / or coding nucleic acid based on a consensus sequence of multiple FN3 domains as described herein, and (b) a preferred and / or pharmaceutically acceptable carrier or diluent.

[0059] This disclosure provides a method for generating a library of protein scaffolds based on fibronectin type III (FN3) repeat proteins, preferably consensus sequences of multiple FN3 domains, more preferably consensus sequences of multiple FN3 domains from human tenascin. The library is formed by creating a series of scaffolds by altering (by mutation) the amino acids or the number of amino acids in the molecule at specific locations in the loop region of a portion of the scaffold. The library can be generated by altering the amino acid composition of a single loop or by simultaneously altering multiple loops or additional locations of the scaffold molecule. The altered loops can be lengthened or shortened accordingly. Such a library can be constructed to include all possible amino acids at each location, or a subset of designed amino acids. Library members can be used for screening by display, such as in vitro or CIS display (DNA, RNA, ribosome display, etc.), yeast, bacterial, and phage display.

[0060] The protein scaffolds of this disclosure offer enhanced biophysical properties, such as stability under reducing conditions and solubility at high concentrations, and can be expressed and folded in prokaryotes such as E. coli, eukaryotes such as yeast, and in vitro transcription / translation systems such as rabbit reticulocyte lysate systems.

[0061] This disclosure provides a method for generating scaffold molecules that bind to a specific target by panning a scaffold library of the present invention with a target and detecting a binder. In other relevant embodiments, this disclosure includes screening methods that can be used to generate or affinity mature protein scaffolds having desired activity and capable of binding to a target protein, for example, with a specific affinity. Affinity maturation can be achieved by repeated rounds of mutagenesis and selection using systems such as phage display or in vitro display. Mutagenesis in this process may be the result of site-directed mutagenesis against specific scaffold residues, random mutagenesis by error-prone PCR, DNA shuffling, and / or a combination of these techniques.

[0062] This disclosure provides isolated, recombinant, and / or synthesized protein scaffolds based on consensus sequences of fibronectin type III (FN3) repeat proteins, and includes, but is not limited to, mammalian-derived scaffolds, as well as compositions and coding nucleic acid molecules comprising at least one polynucleotide encoding a protein scaffold based on a consensus FN3 sequence. This disclosure further includes, but is not limited to, methods for preparing and using such nucleic acids and protein scaffolds, including diagnostic and therapeutic compositions, methods, and devices.

[0063] The protein scaffolds of this disclosure offer advantages over conventional therapies, including the ability to be administered locally, orally, or across the blood-brain barrier; the ability to be engineered into bispecific or tandem molecules that bind to multiple targets or multiple epitopes of the same target; the ability to express proteins in E. coli, enabling increased protein expression as a resource compared to mammalian cell expression capacity; the ability to be conjugated with drugs, polymers, and probes; the ability to be formulated at high concentrations; and the ability of molecules to effectively penetrate diseased tissues and tumors.

[0064] Furthermore, protein scaffolds possess many antibody properties related to their folds, mimicking the variable regions of antibodies. This adaptation allows FN3 loops to be exposed in a similar manner to antibody complementarity-determining regions (CDRs). They should be able to bind to cellular targets, and the loops can improve specific binding or association properties, for example, by altering affinity maturation.

[0065] Three of the six loops of the protein scaffold of this disclosure topologically correspond to the antibody complementarity-determining regions (CDR1-3), i.e., the antigen-binding regions, while the remaining three loops are exposed by means similar to antibody CDRs. These loops span residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81, or their vicinity, of SEQ ID NO: 13. Preferably, the loop regions at residues 22-28, 51-54, and 75-81, or their vicinity, are modified for binding specificity and affinity. One or more of these loop regions are randomized together with other loop regions and / or other chains that maintain their sequences as a skeletal structure, resulting in a library from which potent binding agents can be selected from a library having high affinity for a particular protein target. One or more of the loop regions can interact with the target protein in a manner similar to antibody CDR interactions with the protein.

[0066] The basis of this disclosure may include antibody mimics.

[0067] The term “antibody mimetic” is intended to describe organic compounds that specifically bind to a target sequence and have a structure distinct from naturally occurring antibodies. Antibody mimetic may include proteins, nucleic acids, or small molecules. The target sequences to which the antibody mimetic in this disclosure specifically bind may be antigens. Antibody mimetic may offer properties superior to antibodies, including, but not limited to, superior solubility, tissue penetration, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower manufacturing costs. Exemplary antibody mimetic examples include, but are not limited to, afibodies, afrin, afimer, afitin, alphabodies, antikalin, and abimers (also known as avidity multimers), DARPin (engineered ankyrin repeat protein), Fynomer, Kunitz domain peptides, and monobodies.

[0068] The affibody molecules of this disclosure include one or more alpha-helices that are not crosslinked with disulfide bridges, or include a protein scaffold consisting of such alpha-helices. Preferably, the affibody molecules of this disclosure include or consist of three alpha-helices. For example, the antibody molecule of this disclosure may include an immunoglobulin-binding domain. The affibody molecules of this disclosure may include the Z domain of protein A.

[0069] The affilin molecules of this disclosure include a protein scaffold produced, for example, by modifying exposed amino acids in either gamma B crystallin or ubiquitin. The affilin molecules functionally mimic the affinity of an antibody to an antigen, but do not structurally mimic an antibody. In any protein scaffold used to construct an affilin, any amino acid in a properly folded protein molecule that is accessible to a solvent or potential binding partner is considered an exposed amino acid. One or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.

[0070] The affimer molecules of this disclosure include a protein scaffold containing a highly stable protein that has been genetically engineered to present a peptide loop that yields a high affinity binding site for a specific target sequence. Exemplary affimer molecules of this disclosure include a protein scaffold based on a cystatin protein or its tertiary structure. Exemplary affimer molecules of this disclosure may share a common tertiary structure that includes an alpha helix situated on an antiparallel beta sheet.

[0071] The afitin molecules of this disclosure include an artificial protein scaffold, the structure of which may be derived, for example, from a DNA-binding protein (e.g., DNA-binding protein Sac7d). The afitins of this disclosure selectively bind to a target sequence, which may be all or part of an antigen. Exemplary afitins of this disclosure are produced by randomizing one or more amino acid sequences on the binding surface of a DNA-binding protein and subjecting the resulting protein to ribosome display and selection. The target sequences of the afitins of this disclosure may be found, for example, within a genome or on the surface of a peptide, protein, virus, or bacterium. In certain embodiments of this disclosure, the afitin molecules may be used as specific inhibitors of enzymes. The afitin molecules of this disclosure may include a thermostable protein or a derivative thereof.

[0072] The alpha-body molecules of this disclosure may be referred to as cell-penetrating alpha-bodies (CPABs). The alpha-body molecules of this disclosure contain small proteins (typically less than 10 kDa) that bind to a variety of target sequences (including antigens). The alpha-body molecules can reach and bind to intracellular target sequences. Structurally, the alpha-body molecules of this disclosure contain artificial sequences that form a single-stranded alpha-helix (similar to a naturally occurring coiled-coil structure). The alpha-body molecules of this disclosure may contain a protein scaffold containing one or more amino acids modified to specifically bind to a target protein. Regardless of the molecular binding specificity, the alpha-body molecules of this disclosure maintain correct folding and thermal stability.

[0073] The antikalin molecules of this disclosure include artificial proteins that bind to a target sequence or site in either a protein or a small molecule. The antikalin molecules of this disclosure may include artificial proteins derived from human lipocalin. The antikalin molecules of this disclosure can be used, for example, as a substitute for a monoclonal antibody or a fragment thereof. The antikalin molecules may exhibit superior tissue permeability and thermal stability compared to a monoclonal antibody or a fragment thereof. An exemplary antikalin molecule of this disclosure may contain about 180 amino acids with a mass of about 20 kDa. Structurally, the antikalin molecules of this disclosure include a barrel structure containing pairwise antiparallel beta chains with connected loops and alpha-helices. In a preferred embodiment, the antikalin molecule of this disclosure includes a barrel structure containing eight pairwise antiparallel beta chains with connected loops and alpha-helices.

[0074] The avimer molecules of this disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). The avimers of this disclosure may recognize multiple binding sites within the same target or within different targets. When the avimer of this disclosure recognizes more than one target, it mimics the function of a bispecific antibody. Each artificial protein avimer may contain two or more peptide sequences of approximately 30-35 amino acids. These peptides may be linked via one or more linker peptides. One or more amino acid sequences of the peptides in the avimer may be derived from the A domain of a membrane receptor. The avimers have a robust structure that may optionally contain disulfide bonds and / or calcium. The avimers of this disclosure may exhibit higher thermal stability compared to antibodies.

[0075] The DARPins (engineered ankyrin repeat proteins) of this disclosure include genetically modified proteins, recombinant proteins, or chimeric proteins that have high specificity and high affinity for a target sequence. In certain embodiments, the DARPins of this disclosure are derived from an ankyrin protein and optionally include at least three repeat motifs (also referred to as repeat structural units) of the ankyrin protein. The ankyrin protein mediates high-affinity protein-protein interactions. The DARPins of this disclosure include a large target interaction surface.

[0076] The Fynomer of this disclosure comprises a small binding protein (approximately 7 kDa) derived from a human Fyn SH3 domain and genetically engineered to bind to target sequences and molecules with the same affinity and specificity as the antibody.

[0077] The Kunitz domain peptides of this disclosure include a protein scaffold containing a Kunitz domain. The Kunitz domain contains an active site for inhibiting protease activity. Structurally, the Kunitz domains of this disclosure contain a disulfide-rich alpha+beta fold. This structure is exemplified by bovine pancreatic trypsin inhibitors. The Kunitz domain peptides recognize specific protein structures and act as competitive protease inhibitors. The Kunitz domains of this disclosure may contain ecalantide (derived from human lipoprotein-associated coagulation inhibitors (LACIs)).

[0078] The monobodies of this disclosure are small proteins comparable in size to single-chain antibodies (containing approximately 94 amino acids and having a mass of approximately 10 kDa). These genetically engineered proteins specifically bind to target sequences containing antigens. The monobodies of this disclosure may specifically target one or more distinctly different proteins or target sequences. In a preferred embodiment, the monobodies of this disclosure include a protein scaffold that mimics the structure of human fibronectin, and more preferably, the structure of the 10th extracellular type III domain of fibronectin. The 10th extracellular type III domain of fibronectin, and its monobodies, contain seven beta sheets and three exposed loops forming a barrel on each side corresponding to the three complementarity-determining regions (CDRs) of the antibody. In contrast to the structure of the variable domain of the antibody, the monobodies lack binding sites for metal ions and a central disulfide bond. Multispecific monobodies can be optimized by modifying loops BC and FG. The monobodies of this disclosure may contain adnectin.

[0079] Therefore, the method may include administering an effective amount of a composition or pharmaceutical composition containing at least one scaffold protein to cells, tissues, organs, animals, or patients who require such modulation, treatment, relief, prevention, or reduction of symptoms, effects, or mechanisms. The effective amount may include an amount of about 0.001 to 500 mg / kg per single (e.g., bolus), multiple, or consecutive doses, or an amount that achieves a serum concentration of 0.01 to 5000 μg / ml per single, multiple, or consecutive doses, or any effective range or value within that range, which is performed and determined using methods described herein or known in the related art.

[0080] Production and generation of scaffold proteins At least one of the scaffold proteins of this disclosure can be selectively produced by cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells, as is well known in the art. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); and Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0081] Amino acids from scaffold proteins may be modified, added, and / or deleted, as is known in the art, to reduce immunogenicity, or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility, or any other desirable properties.

[0082] Optionally, scaffold proteins can be manipulated while retaining high affinity for antigens and other desirable biological properties. To achieve this goal, scaffold proteins can be optionally prepared by analytical processes of the parent sequence and various conceptually manipulated products using three-dimensional models of the parent sequence and the manipulated sequence. Three-dimensional models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional structures of selected candidate sequences, and their potential immunogenicity can be measured (e.g., the Immunofilter program from Xencor, Inc. (Monrovia, Calif.)). Examination of these displays allows for the analysis of possible roles of residues in the function of the candidate sequence, i.e., the analysis of residues that affect the ability of the candidate scaffold protein to bind to its antigen. Thus, residues can be selected and combined from the parent sequence and reference sequence so that desired characteristics, such as affinity for the target antigen, are achieved. Alternatively, or in addition to the above procedure, other suitable manipulation methods may be used.

[0083] Screening of scaffold proteins Screening of protein scaffolds for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro displays. This method involves screening large assemblies of peptides for individual members having the desired function or structure. Displayed nucleotide or peptide sequences may be 3 to 5,000 or more nucleotides or amino acids long, often 5 to 100 amino acids long, and often about 8 to 25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves displaying peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a specific displayed peptide sequence. Such methods are described in PCT Patent Publications 91 / 17271, 91 / 18980, 91 / 19818, and 93 / 08278.

[0084] Other systems for generating peptide libraries include both in vitro chemical synthesis and recombinant methods. See PCT Patent Publications 92 / 05258, 92 / 14843, and 96 / 19256. See also U.S. Patents 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from suppliers such as Invitrogen (Carlsbad, Calif.) and Cambridge Antibody Technologies (Cambridgeshire, UK). For example, U.S. patents No. 4,704,692, No. 4,939,666, No. 4,946,778, No. 5,260,203, No. 5,455,030, No. 5,518,889, No. 5,534,621, No. 5,656,730, No. 5,763,733, No. 5,767,260, and No. 5,856,456, which were transferred to Enzon; ​​No. 5,223,409, No. 5,403,484, No. 5,571,698, and No. 5,837,500, which were transferred to Dyax; No. 5,427,908 and No. 5,580,717, which were transferred to Affymax; and Cambridge Antibody. See also No. 5,885,793, transferred to Technologies; No. 5,750,373, transferred to Genentech; and Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, and 5,698,417, transferred to Xoma, Colligan, Ausubel, or Sambrook.

[0085] The protein scaffolds disclosed herein can bind to human or other mammalian proteins with a wide range of affinity (KD). In a preferred embodiment, at least one protein scaffold of the disclosure can optionally bind to a target protein with high affinity, as determined by surface plasmon resonance or Kinexa method, as practiced by those skilled in the art, with a KD of about 10⁻⁷ M or less, or any range or value between these, for example, 0.1 to 9.9 (or any range or value between these) × 10⁻⁸, 10⁻⁹, 10⁻¹, 10⁻¹¹, 10⁻¹², 10⁻¹³, ​​10⁻¹⁴, 10⁻¹⁵, etc., but not limited to these, or any range or value between these.

[0086] The affinity or avidity of a protein scaffold to an antigen can be experimentally determined using any suitable method. (See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein). The measured affinity of a particular protein scaffold-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, the measurement of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) is preferably performed using standardized solutions of the protein scaffold and antigen, as well as standardized buffers such as those described herein.

[0087] Competition assays can be performed using the protein scaffolds of this disclosure to determine which proteins, antibodies, and other antagonists compete for binding to target proteins having the protein scaffolds of this disclosure and / or share epitope regions. These assays, readily known to those skilled in the art, evaluate competition between antagonists or ligands for a limited number of binding sites on the protein. Proteins and / or antibodies are immobilized or insolubilized before and after competition, and the sample bound to the target protein is separated from the unbound sample, for example, by decantation (if the protein / antibody is insolubilized beforehand) or centrifugation (if the protein / antibody precipitates after the competition reaction). Competitive binding can also be determined by whether the function of the target protein changes due to binding to or lack of binding to the protein scaffold, for example, whether the protein scaffold molecule inhibits or enhances the enzyme activity of the label, for example. As is well known in the art, ELISA and other functional assays can be used.

[0088] nucleic acid molecule The nucleic acid molecules of this disclosure that encode protein scaffolds may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand, also known as a sense strand, or a non-coding strand, also referred to as an antisense strand.

[0089] The isolated nucleic acid molecules of this disclosure may include nucleic acid molecules comprising an open reading frame (ORF), optionally comprising one or more introns, e.g., at least one identified portion of at least one protein scaffold, a nucleic acid molecule comprising a coding sequence of a protein scaffold or loop region that binds to a target protein, and a nucleic acid molecule comprising a nucleotide sequence substantially different from the above but still encoding a protein scaffold described herein and / or known in the art for the degenerate nature of the genetic code. Of course, the genetic codes are well known in the art. Therefore, generating degenerate nucleic acid variants encoding a particular protein scaffold of the present invention is commonplace for those skilled in the art. For example, Ausubel, et al., refer to the above, such nucleic acid variants are included in the present invention.

[0090] As described herein, nucleic acid molecules of this disclosure that encode a protein scaffold may include, but are not limited to, sequences encoding additional amino acids, such as sequences encoding an amino acid sequence of a protein scaffold fragment; sequences encoding the entire protein scaffold or a portion thereof; sequences encoding the protein scaffold, fragment, or a portion thereof, along with additional non-coding sequences, including but not limited to transcribed untranslated sequences and non-coding 5' and 3' sequences, such as polyadenylation signals (e.g., ribosome binding and stability of mRNA), which play a role in transcription, which is mRNA processing including splicing, and at least one intron; and additional coding sequences encoding additional amino acids, such as sequences that provide additional functionality. Thus, sequences encoding a protein scaffold can be fused to marker sequences, such as sequences encoding peptides that facilitate the purification of fusion protein scaffolds, including protein scaffold fragments or portions thereof.

[0091] Polynucleotides that selectively hybridize to the polynucleotides described herein This disclosure provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposit library. In some embodiments, the polynucleotides are cDNA isolated from a human or mammalian nucleic acid library, or otherwise complementary genomic or cDNA sequences.

[0092] Preferably, the cDNA library contains at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or moderately stringent hybridization conditions are not exclusive but are typically used for sequences with reduced sequence identity compared to complementary sequences. Moderate and highly stringent conditions can be used selectively for sequences with higher identity. Low stringent conditions allow for selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0093] Optionally, the polynucleotides of the present invention encode at least a portion of the protein scaffold encoded by the polynucleotides described herein. The polynucleotides of the present invention encompass nucleic acid sequences that can be used for selective hybridization with the polynucleotides encoding the protein scaffold of the present invention. See, for example, Ausubel, above, and Colligan, above, each of which is incorporated herein by reference in whole.

[0094] Nucleic acid construction The isolated nucleic acids of this disclosure can be prepared using (a) recombinant methods, (b) synthesis techniques, (c) purification techniques, and / or (d) combinations thereof, as are well known in the art.

[0095] Nucleic acids may conveniently contain sequences in addition to the polynucleotides of this disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of polynucleotides. Alternatively, a translatable sequence can be inserted to aid in the isolation of translated polynucleotides of this disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of this disclosure. The nucleic acids of this disclosure, excluding the coding sequence, are optionally vectors, adapters, or linkers for cloning and / or expression of the polynucleotides of this disclosure.

[0096] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, aid in the isolation of polynucleotides, or improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, for example, Ausubel, above; or Sambrook, above).

[0097] Recombination methods for constructing nucleic acids The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some embodiments, desired sequences in cDNA or genomic DNA libraries are identified using oligonucleotide probes that selectively hybridize to the polynucleotides of the present invention under stringent conditions. The isolation of RNA, as well as the construction of cDNA and genomic libraries, are well known to those skilled in the art (see, for example, Ausubel, above; or Sambrook, above).

[0098] Nucleic acid screening and isolation methods cDNA or genomic libraries can be screened using the polynucleotide sequence-based probes of this disclosure. The probes can be hybridized to genomic DNA or cDNA sequences to isolate identical or different homologous genes in vivo. Those skilled in the art will understand that varying degrees of hybridization stringency can be used in an assay, and that either the hybridization medium or the washing medium can be stringent. The stringier the conditions for hybridization, the higher the degree of complementarity between the probe and the target must be for double-strand formation to occur. The degree of stringency can be controlled by one or more of the following: temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization can be conveniently altered by changing the polarity of the reaction solution, for example, through manipulation of the formamide concentration in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies according to the stringency of the hybridization medium and / or washing medium. The degree of complementarity is optimally 100%, or 70–100%, or any range or value in between. However, it should be understood that slight sequence variations within the probe and primer can be compensated for by reducing the stringency of hybridization and / or washing medium.

[0099] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with this disclosure without excessive experimentation, based on the teachings and guidelines presented herein.

[0100] Known methods for DNA or RNA amplification include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Patents No. 4,683,195, 4,683,202, 4,800,159, 4,965,188 (Mullis et al.), 4,795,699, and 4,921,794 (Tabor et al.), 5,142,033 (Innis), 5,122,464 (Wilson et al.), 5,091,310 (Innis), 5,066,584 (Gyllensten et al.), 4,88...). This includes, but is not limited to, U.S. Patent No. 9,818 (Gelfand et al.), No. 4,994,370 (Silver et al.), No. 4,766,067 (Biswas), No. 4,656,134 (Ringold), and RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 (Malek et al.), trademark NASBA), and the full contents of these references are incorporated herein by reference. (See, for example, Ausubel, above; or Sambrook, above.)

[0101] For example, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the polynucleotides and associated genes of this disclosure directly from a genomic DNA or cDNA library. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences encoding proteins to be expressed, to detect the presence of desired mRNA in a sample, to sequence nucleic acids, or to produce nucleic acids for use as probes for other purposes. Examples of techniques sufficient to guide those skilled in the art through in vitro amplification methods can be found in Berger, above, Sambrook, above, and Ausubel, above, as well as U.S. Patent No. 4,683,202 (1987) by Mullis et al., and innis et al., PCR Protocols: A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercial kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[0102] Synthesis methods for constructing nucleic acids The isolated nucleic acids of this disclosure may also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., above). Chemical synthesis generally produces single-stranded oligonucleotides that can be converted to double-stranded DNA by hybridization with complementary sequences or polymerization with DNA polymerase using a single strand as a template. Those skilled in the art will recognize that while the chemical synthesis of DNA may be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligation of shorter sequences.

[0103] Recombinant expression cassette This disclosure further provides recombinant expression cassettes comprising the nucleic acids of the Disclosure. Recombinant expression cassettes can be constructed using nucleic acid sequences of the Disclosure, for example, cDNA or genomic sequences encoding a protein scaffold of the Disclosure, which can be introduced into at least one desired host cell. The recombinant expression cassette would typically contain polynucleotides of the Disclosure operably ligated to transcription initiation regulatory sequences that lead to the transcription of polynucleotides in the intended host cell. Expression of the nucleic acids of the Disclosure can be led using both heterogeneous and non-heterogeneous (i.e., endogenous) promoters.

[0104] In some embodiments, isolated nucleic acids functioning as promoters, enhancers, or other elements can be introduced at appropriate locations (upstream, downstream, or within an intron) of non-heterogeneous forms of the polynucleotides disclosed in order to upregulate or downregulate the expression of the polynucleotides disclosed. For example, an endogenous promoter can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0105] Vectors and host cells This disclosure also relates to vectors containing isolated nucleic acid molecules of this disclosure, host cells genetically engineered with recombinant vectors, and the production of at least one protein scaffold by recombinant technology, as is well known in the art. See, for example, Sambrook et al., and Ausubel et al., respectively, each incorporated herein by reference in whole.

[0106] For example, the PB-EF1a vector can be used. The vector contains the following nucleotide sequence:

[0107] Polynucleotides can be selectively bound to vectors containing selectable markers for replication in a host. Generally, plasmid vectors are introduced into a precipitate, such as calcium phosphate precipitate, or into a complex with charged lipids. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

[0108] The DNA insert should be operablely ligated to an appropriate promoter. The expression construct will further include sites for transcription initiation and termination, and in the transcribed region, ribosome-binding sites for translation. The coding portion of the mature transcript expressed by the construct preferably includes translation beginning with start and stop codons (e.g., UAA, UGA, or UAG) appropriately located at the end of the mRNA to be translated, with UAA and UAG being preferred in expression in mammalian or eukaryotic cells.

[0109] The expression vector preferably, but optionally, includes at least one selectable marker. Such markers include, for example, ampicillin, zeosin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), mycophenolic acid or glutamine synthase (GS, U.S. Patent Nos. 5,122,464, 5,770,359, and 5,827,739) for eukaryotic cell culture, blasticidine (bsd gene), resistance genes, and ampicillin, zeosin (Sh This includes, but is not limited to, the bla gene, puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes (the above patents are incorporated herein by reference in their entirety). Suitable culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be achieved by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, such as Sambrook, Chapters 1-4 and 16-18 above, and Ausubel, Chapters 1, 9, 13, 15, and 16 above.

[0110] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for the isolation of cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure include a group of surface proteins, glycoproteins, or proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the compositions or methods of the present disclosure from cells that have not been modified by the compositions or methods of the present disclosure. Such cell surface markers include, but are not limited to, "cluster of designation" or "classification determinant" proteins (often abbreviated as "CD"), such as cleaved or full-length CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers also include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug21;124(8):1277-87).

[0111] The expression vector will preferably, but optionally, include at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0112] At least one protein scaffold of this disclosure may be expressed in a modified form, such as a fusion protein, and may include not only secretory signals but also further heterogeneous functional regions. For example, additional amino acid regions, particularly charged amino acid regions, can be added to the N-terminus of the protein scaffold to improve stability and survival in host cells during purification or subsequent handling and storage. Alternatively, peptide moieties can be added to the protein scaffold of this disclosure to facilitate purification. Such regions can be removed before the final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, Chapters 17.29-17.42 and 18.1-18.74 above, and Ausubel, Chapters 16, 17, and 18 above.

[0113] Those skilled in the art are familiar with numerous expression systems available for expressing the nucleic acids encoding the proteins of the Disclosure. Alternatively, the nucleic acids of the Disclosure can be expressed in host cells by (manipulating) turning them on in host cells containing endogenous DNA encoding the protein scaffold of the Disclosure. Such methods are well known in the art, for example, as described in U.S. Patents 5,580,734, 5,641,670, 5,733,746, and 5,733,761 (which are incorporated herein by reference in their entirety).

[0114] Cell cultures that serve as useful examples for producing protein scaffolds, specific parts thereof, or mutants are bacterial, yeast, and mammalian cells, which are known in the art. Mammalian cell systems are often in the form of a single layer of cells, but mammalian cell suspensions or bioreactors can also be used. Numerous suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC CRL1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, etc., which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include lymphoid cells such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In a particularly preferred embodiment, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0115] The expression vectors of these cells may include one or more regulatory sequences, including, but not limited to, a replication origin, a promoter (e.g., late or early SV40 promoter, CMV promoter (US Patent No. 5,168,062, 5,385,839), HSV tk promoter, pgk (phosphoglycerin kinase) promoter, EF-1 alpha promoter (US Patent No. 5,266,491), at least one human promoter, enhancer, and / or processing information site (e.g., ribosome binding site, RNA splice site, polyadenylation site (e.g., SV40 large T Ag poly-A addition site)), and a transcription termination sequence. See, for example, Ausubel et al. and Sambrook et al. Other cells useful for the production of nucleic acids or proteins of the present invention are known and / or available from, for example, the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0116] When eukaryotic host cells are used, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). In addition, gene sequences for controlling replication in the host cell can be incorporated into the vector, as is known in the art.

[0117] Purification of protein scaffolds Protein scaffolds can be recovered and purified from recombinant cell cultures by known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Chapters 1, 4, 6, 8, 9, and 10 of Colligan, *Current Protocols in Immunology*, or *Current Protocols in Protein Science*, John Wiley & Sons, NY, NY, (1997-2001), which are incorporated herein by reference, for example.

[0118] The protein scaffolds of this disclosure include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, E. coli, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production procedure, the protein scaffolds of this disclosure may or may not be glycosylated. Such methods are described in many standard experimental manuals, such as Sambrook, Sections 17.37-17.42 above; Ausubel, Chapters 10, 12, 13, 16, 18, and 20 above; Colligan, Protein Science, Chapters 12-14 above above; all of which are incorporated herein by reference.

[0119] Amino acid code The amino acids constituting the protein scaffolds of this disclosure are often abbreviated. Amino acid names can be identified by their one-letter code, three-letter code, name, or by three nucleotide codons, as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). The protein scaffolds of this disclosure may include substitutions, deletions, or additions of one or more amino acids, either naturally occurring or through human manipulation, as specified herein. Amino acids in the protein scaffolds of this disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, above, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue of the molecule. Next, the resulting mutant molecules are tested for biological activities, including but not limited to neutralizing activity. Sites crucial for protein scaffold binding can be identified by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J.Mol.Biol.224:899-904 (1992) and de Vos, et al., Science255:306-312 (1992)).

[0120] As those skilled in the art will understand, the present invention comprises at least one biologically active protein scaffold of the present disclosure. The biologically active protein scaffold has a specific activity of at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 99% or higher, of the specific activity of a natural (non-synthetic), endogenous, or related and known protein. Methods for assaying and quantifying measures of enzyme activity and substrate specificity are well known to those skilled in the art.

[0121] In another embodiment, the disclosure relates to protein scaffolds and fragments described herein that are modified by covalent bonding of organic moieties. Such modifications can produce protein scaffold fragments having improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moieties may be linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups. In certain embodiments, the hydrophilic polymer groups may have a molecular weight of about 800 to about 120,000 daltons and may be polyalkane glycols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymers, amino acid polymers, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester groups may contain about 8 to about 40 carbon atoms.

[0122] The modified protein scaffolds and fragments of this disclosure may include one or more organic moieties directly or indirectly covalently bound to an antibody. Each organic moiety bound to a protein scaffold or fragment of this disclosure may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term “fatty acid” includes monocarboxylic acids and dicarboxylic acids. “Hydrophilic polymer group” as used herein refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Therefore, protein scaffolds modified by covalent bonding of polylysine are encompassed by this disclosure. Suitable hydrophilic polymers for modifying the protein scaffolds of this disclosure can be linear or branched and may include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers for modifying the protein scaffolds of this disclosure have a molecular weight of about 800 to about 150,000 daltons as separate molecular entities. For example, PEG5000 and PEG20000 (where the subscript is the average molecular weight of the polymer in daltons) can be used. The hydrophilic polymer groups can be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by preferred methods. For example, polymers containing amine groups can be coupled to carboxylates of fatty acids or fatty acid esters, and activated carboxylates on fatty acids or fatty acid esters (e.g., activated with N,N-carbonyldiimidazole) can be coupled to hydroxyl groups on the polymer.

[0123] Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of this disclosure may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffolds of this disclosure include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-Δ9-octadecanoate (C18, oleate), total cis-Δ5,8,11,14-eicosatetraenoate (C20, arachidonic acid), octanedioic acid, tetradecanediic acid, octadecanediic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6, carbon atoms.

[0124] Modified protein scaffolds and fragments can be prepared using preferred methods, such as by reaction with one or more modifiers. As used herein, the term “modifier” refers to preferred organic groups containing activating groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters). An “activating group” is a chemical moiety or functional group that can react with a second chemical group under suitable conditions, thereby forming a covalent bond between the modifier and the second chemical group. For example, amine-reactive activating groups include electrophiles such as tosylates, mesylates, halo(chloro, bromo, fluoro, iodine), and N-hydroxysuccinimidyl esters (NHS). Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloryl, pyridyl disulfide, and 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol). Aldehyde functional groups can be bonded to amine-containing molecules or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Preferred methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be bonded directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or through linker moieties, such as divalent C1-C12 groups in which one or more carbon atoms can be substituted with heteroatoms such as oxygen, nitrogen, or sulfur. Preferred linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. Modifiers containing a linker moiety can be generated, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be bonded to another carboxylate as described, or it can be reacted with maleic anhydride, and the resulting product can be cyclized to produce an activated maleimide derivative of the fatty acid. (See, for example, Thompson, et al., WO92 / 16221, the entire teaching of which is incorporated herein by reference.)

[0125] The modified protein scaffolds of this disclosure can be generated by reacting a protein scaffold or fragment with a modifier. For example, the organic portion can be attached to the protein scaffold by a non-site-specific means using an amine-reactive modifier, such as an NHS ester of PEG. Modified protein scaffolds and fragments containing organic moieties that bind to specific sites of the protein scaffolds of this disclosure can be prepared using preferred methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992), Werlen et al., Bioconjugate Chem., 5:411-417 (1994), Kumaran et al., Protein Sci. 6(10):2233-2241 (1997), Itoh et al., Bioorg. Chem., 24(1):59-68 (1996), Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and preferred methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0126] Protein scaffold composition containing further therapeutic active ingredients The protein scaffold compounds, compositions, or combinations of this disclosure may further include at least one of any suitable auxiliary agents, including but not limited to diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, and adjuvants. Pharmaceutically acceptable auxiliary agents are preferred. Non-limited examples of such sterile solutions and methods for their preparation are well known in the art, for example, but not limited to Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers may be well known in the art or can be systematically selected for their suitability to the administration, solubility, and / or stability of the protein scaffolds, fragments, or variant compositions described herein.

[0127] Useful pharmaceutical excipients and additives for this composition include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, derivatized sugars such as alditol, aldonic acid, and esterified sugars, and polysaccharides or sugar polymers), and may exist alone or in combination, or may constitute 1 to 99.99% by weight or volume. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acid / protein components that can also function in terms of buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. One preferred amino acid is glycine.

[0128] Suitable carbohydrate excipients for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.

[0129] Protein scaffold compositions may also contain buffers or pH adjusters, typically the buffers being salts prepared from organic acids or bases. Typical buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, tris, tromethamine hydrochloride, or phosphate buffers. Organic acid salts such as citrates are preferred buffers for use in this composition.

[0130] In addition, the protein scaffold composition of the present invention may contain polymer excipients / additives, such as polyvinylpyrrolidone, ficol (high molecular weight sugar), dextrose (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antibacterial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN20" and "TWEEN80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0131] These and additional known pharmaceutically acceptable excipients and / or additives suitable for use in protein scaffolds, partials, or variant compositions according to the present invention are known in the art and are listed, for example, in “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, (1995) and “Physician's Desk Reference”, 52nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are incorporated herein by reference in their entirety. Preferred carrier or excipient materials are carbohydrates (e.g., sugars and alditols) and buffers (e.g., citrates), or polymers. Exemplary carrier molecules include mucopolysaccharides and hyaluronic acid, which may be useful for intra-articular delivery.

[0132] Isolation of T cells from leukocyte apheresis products Leukocyte apheresis products or blood can be collected from subjects in a clinical setting using a closed system and standard methods (e.g., the COBE Spectra Apheresis System). Preferably, the products are collected in a standard leukocyte apheresis collection bag according to standard hospital or institution leukocyte apheresis procedures. For example, in a preferred embodiment of the method of this disclosure, no additional anticoagulants or blood additives (such as heparin) are included in addition to those typically used during leukocyte apheresis.

[0133] Alternatively, leukocytes (WBCs) / peripheral blood mononuclear cells (PBMCs) (using Biosafe Sepax2 (closed / automated)) or T cells (using CliniMACS® Prodigy (closed / automated)) can be isolated directly from whole blood. However, in certain subjects (e.g., subjects diagnosed with and / or treated for cancer), the yield of WBCs / PBMCs may be significantly lower when isolated from whole blood than when isolated by leukocyte apheresis.

[0134] The leukocyte apheresis procedure and / or the direct cell isolation procedure can both be used for any subject of this disclosure.

[0135] Leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions should be filled into shielded containers and maintained at a controlled room temperature (+19°C to +25°C) in accordance with standard hospital or institutional blood collection procedures approved for use in clinical protocols. Leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions should not be refrigerated.

[0136] The cell concentration of leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions during transport is 0.2 × 10⁴ per mL. 9 The mixture should not exceed the individual cell level. Vigorous mixing of leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions should be avoided.

[0137] If leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions must be stored, for example, overnight, they must be maintained at a controlled room temperature (same as above). During storage, the concentration of leukocyte transfer products, blood, WBC / PBMC compositions, and / or T cell compositions should be 0.2 × 10⁶ per mL. 9 It must not exceed the limits of an individual cell.

[0138] Preferably, the cells of the leukocyte apheresis product, blood, WBC / PBMC composition, and / or T cell composition should be stored in the patient's own plasma. In certain embodiments, the cell concentration of the leukocyte apheresis product, blood, WBC / PBMC composition, and / or T cell composition is 0.2 × 10⁶ per mL. 9 If the concentration is higher than that of individual cells, the product should be diluted with the patient's own plasma.

[0139] Preferably, the leukocyte apheresis product, blood, WBC / PBMC composition, and / or T cell composition should not be more than 24 hours old when the labeling and separation procedure is initiated. The leukocyte apheresis product, blood, WBC / PBMC composition, and / or T cell composition can be processed and / or prepared for cell labeling using a closed and / or automated system (e.g., CliniMACS Prodigy).

[0140] The automated system may, in some cases, perform further buffy coat isolation by ficolation and / or washing of cell products (e.g., leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions).

[0141] Using closed and / or automated systems, cells can be prepared and labeled for T cell isolation (e.g., from leukocyte apheresis products, blood, WBC / PBMC compositions, and / or T cell compositions).

[0142] While WBCs / PBMCs may be directly nucleofected (which is easy and saves additional steps), the method of this disclosure may include the step of first isolating T cells before nucleofection. The easy strategy of directly nucleofecting PBMCs requires selective augmentation of CAR+ cells mediated by CAR signaling, which in itself has been shown to be an inferior augmentation method that directly reduces the in vivo efficiency of the product by functionally depleting T cells. The product may be a heterogeneous composition of CAR+ cells including T cells, NK cells, NKT cells, monocytes, or any combination thereof, which increases patient-to-patient product variability and makes drug administration and CRS management more difficult. Since T cells are considered to be the major effectors in tumor suppression and death, isolating T cells for the production of the product of the self may yield significant advantages over other more heterogeneous compositions.

[0143] T cells can be isolated directly by enriching or depleting labeled cells using a unidirectional labeling procedure, or indirectly by a two-step labeling procedure. According to the specific enrichment strategy of this disclosure, T cells can be collected in a cell collection bag, and unlabeled cells (non-target cells) can be collected in a negative fraction bag. In contrast to the enrichment strategy of this disclosure, unlabeled cells (target cells) are collected in a cell collection bag, and labeled cells (non-target cells) are collected in a negative fraction bag or a non-target cell bag, respectively. Selective reagents may include, but are not limited to, antibody-coated beads. Antibody-coated beads may be removed before the modification and / or augmentation step, or may be retained on the cells before the modification and / or augmentation step. One or more of the following non-limiting examples of cell markers can be used to isolate T cells: CD3, CD4, CD8, CD25, antibiotin, CD1c, CD3 / CD19, CD3 / CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN-gamma, TCR alpha / beta, and / or any combination thereof. A method for isolating T cells may include one or more reagents that specifically bind to and / or detectably label one or more of the following non-limiting examples of cell markers that can be used for T cell isolation: CD3, CD4, CD8, CD25, antibiotin, CD1c, CD3 / CD19, CD3 / CD56, CD14, CD19, CD34, CD45RA, CD56, CD62L, CD133, CD137, CD271, CD304, IFN-gamma, TCR alpha / beta, and / or any combination thereof. These reagents may or may not be of Good Manufacturing Practice (GMP) grade. Reagents may include, but are not limited to, Thermo DynaBeads and Miltenyi CliniMACS products. The method for isolating T cells of this disclosure may include multiple repetitions of the labeling and / or isolation step.At any point in the method for isolating T cells of the present disclosure, undesirable cells and / or cell types may be depleted from the T cell product composition of the present disclosure by positive or negative selection of undesirable cells and / or cell types. The T cell product composition of the present disclosure may contain further cell types capable of expressing CD4, CD8, and / or other T cell markers.

[0144] The present disclosure of the method for nucleofection of T cells may exclude the step of T cell isolation, for example, by a process of nucleofection of a population or composition of WBCs / PBMCs that includes an isolation step or a selective augmentation step via TCR signaling after nucleofection.

[0145] Certain cell populations may be depleted by bioselection or negative selection before or after T cell enrichment and / or classification. Examples of cell compositions that may be depleted from cell product compositions may include myeloid cells, CD25+ regulated T cells (T Regs), dendritic cells, macrophages, erythrocytes, mast cells, gamma-delta T cells, natural killer (NK) cells, natural killer (NK)-like cells (e.g., cytokine-induced killer (CIK) cells), induced natural killer (iNK) T cells, NK T cells, B cells, or any combination thereof.

[0146] The T cell product compositions of this disclosure may comprise CD4+ and CD8+ T cells. CD4+ and CD8+ T cells can be isolated into separate collection bags during isolation or selection procedures. CD4+ and CD8+ T cells can be further processed separately or processed after being reconstituted (combined into the same composition) in specific ratios.

[0147] The specific ratios in which CD4+ T cells and CD8+ T cells can be reconstituted may depend on the type and effectiveness of the augmentation technique used, the cell medium, and / or the growth conditions utilized for the augmentation of the T cell product composition. Examples of possible CD4+:CD8+ ratios include, but are not limited to, 50%:50%, 60%:40%, 40%:60%, 75%:25%, and 25%:75%.

[0148] CD8+ T cells exhibit a potent ability to kill tumor cells, while CD4+ T cells provide many of the cytokines necessary to support the proliferative capacity and function of CD8+ T cells. Since T cells isolated from a normal donor are predominantly CD4+, the composition of the T cell product is artificially adjusted in vitro with respect to the CD4+:CD8+ ratio, improving the ratio of CD4+ T cells to CD8+ T cells that would otherwise be present in vivo. The optimized ratio can also be used for ex vivo augmentation of the autologous T cell product composition. Given the artificially adjusted CD4+:CD8+ ratio of the T cell product composition, it is important to note that the product composition of this disclosure may differ significantly from any naturally occurring population of T cells and may offer significantly greater advantages.

[0149] A preferred method for T cell isolation may involve a negative selection strategy that produces untouched pan-T cells, meaning that the resulting T cell composition contains T cells that are unmanipulated and include naturally occurring T cell types / ratios.

[0150] Reagents that can be used for positive or negative selection include, but are not limited to, magnetic cell isolation beads. Magnetic cell isolation beads may be removed or depleted from a selected population of CD4+ T cells, CD8+ T cells, or a mixed population of both CD4+ and CD8+ T cells before performing the next step of the T cell isolation method of this disclosure.

[0151] T cell compositions and T cell product compositions may be prepared for cryopreservation, chilling in standard T cell culture media, and / or genetic modification.

[0152] T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any part thereof can be cryopreserved using standard cryopreservation methods optimized for storing and restoring human cells with high recovery, viability, phenotype, and / or functional capacity. Commercially available cryopreservation media and / or protocols can be used. The cryopreservation methods of this disclosure may include DMSO-free cryopreserving agents that reduce cryo-related toxicity (e.g., CryoSOfree® DMSO-free cryopreservation medium).

[0153] T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any portion thereof may be stored in a culture medium. The T cell culture medium of this disclosure can be optimized for cell storage, cell genetic modification, cell phenotype, and / or cell growth. The T cell culture medium of this disclosure may contain one or more antibiotics. Since the inclusion of antibiotics in the cell culture medium may reduce transfection efficiency and / or cell yield after genetic modification by nucleofection, specific antibiotics (or combinations thereof) and their respective concentrations may be modified for optimal transfection efficiency and / or cell yield after genetic modification by nucleofection.

[0154] The T cell culture media of this disclosure may contain serum, and furthermore, serum compositions and concentrates may be modified for optimal cell outcomes. Human AB serum is intended for use in the T cell culture media of this disclosure, but FBS / FCS is preferred over FBS / FCS for T cell culture because it can induce heterologous proteins. The serum may be isolated from the blood of the subject from whom the T cell composition in the culture is intended for administration, and therefore the T cell culture media of this disclosure may contain the subject's own serum. Serum-free media or serum substitutes may also be used in the T cell culture media of this disclosure. In certain embodiments of the T cell culture media and methods of this disclosure, serum-free media or serum substitutes may provide advantages over supplementing the medium with heterologous serum, including, but not limited to, healthier cells that have better viability, are nucleofected more efficiently, exhibit better viability after nucleofection, exhibit a more desirable cell phenotype, and / or exhibit better / faster growth when augmentation techniques are added.

[0155] T cell culture media may include commercially available cell growth media. Exemplary commercially available cell growth media include, but are not limited to, PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC medium, CTS OpTimizer T cell growth medium SFM, TexMACS medium, PRIME-XV T cell growth medium, ImmunoCult-XF T cell growth medium, or any combination thereof.

[0156] T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any part thereof can be prepared for genetic modification. Preparation of T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any part thereof for genetic modification may include cell washing and / or resuspension in a desired nucleofection buffer. Cryopreserved T cell compositions can be thawed and prepared for genetic modification by nucleofection. Cryopreserved cells can be thawed according to standard or known protocols. The thawing and preparation of cryopreserved cells can be optimized to produce cells with better viability, higher efficiency of nucleofection, better viability after nucleofection, a more desirable cell phenotype, and / or better / faster growth when augmentation techniques are added. For example, Grifols Albutein (25% human albumin) can be used in the thawing and / or preparation process.

[0157] Genetic modification of autologous T cell product composition T cell compositions, T cell product compositions, unstimulated T cell compositions, resting T cell compositions, or any part thereof can be genetically modified using nucleofection strategies such as electroporation. By optimizing the total number of cells nucleofected, the total volume of the nucleofection reaction, and the precise timing of sample preparation, it is possible to produce cells that have better viability, are nucleofected with higher efficiency, exhibit better viability after nucleofection, have a more desirable cell phenotype, and / or exhibit better / faster growth when growth techniques are added.

[0158] Nucleofection and / or electroporation can be achieved using, for example, Lonza Amaxa, MaxCyte PulseAgile, Harvard Apparatus BTX, and / or Invitrogen Neon. Nonmetallic electrode systems, including but not limited to plastic polymer electrodes, may be preferred for nucleofection.

[0159] Prior to genetic modification by nucleofection, the T cell composition, T cell product composition, unstimulated T cell composition, resting T cell composition, or any portion thereof may be resuspended in a nucleofection buffer. The nucleofection buffer of this disclosure includes commercially available nucleofection buffers. The nucleofection buffer of this disclosure can be optimized to produce cells that have better viability, are nucleofected more efficiently, exhibit better viability after nucleofection, have a more desirable cell phenotype, and / or exhibit better / faster growth when growth techniques are added. The nucleofection buffer of this disclosure may include, but is not limited to, PBS, HBSS, OptiMEM, BTXpres, Amaxa Nucleofector, human T cell nucleofection buffer, and any combination thereof. The nucleofection buffers of this disclosure contain one or more co-factors and can produce cells that have superior viability, are nucleofected with higher efficiency, exhibit superior viability after nucleofection, have a more desirable cell phenotype, and / or exhibit superior / faster growth when growth techniques are added. Exemplary co-factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins, and any combination thereof.Exemplary cytokines, chemokines, and interleukins include IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANKL, and any combination thereof are included but not limited to these. Exemplary co-factors include but are not limited to salts, inorganic substances, metabolites, or any combination thereof. Exemplary salts, inorganics, and metabolites include, but are not limited to, HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum substitutes, antibiotics, pH adjusters, Earl's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCl, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof.Exemplary co-enhancing factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC medium, CTS OpTimizer T cell growth medium SFM, TexMACS medium, PRIME-XV T cell growth medium, ImmunoCult-XF T cell growth medium, and any combination thereof. Exemplary co-enhancing factors include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signaling, apoptotic pathways, and combinations thereof. Exemplary inhibitors include, but are not limited to, inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, and any combination thereof. Exemplary auxiliary factors include, but are not limited to, reagents that modify or stabilize one or more nucleic acids to enhance cell delivery, enhance nuclear delivery or transport, enhance assisted transport of nucleic acids to the nucleus, enhance the degradation of nucleic acids on chromosomes, and / or reduce DNA-mediated toxicity. Exemplary reagents that modify or stabilize one or more nucleic acids include, but are not limited to, pH adjusters, DNA-binding proteins, lipids, phospholipids, CaPO4, net-neutral charge DNA-binding peptides with or without NLS sequences, TREX1 enzymes, and any combination thereof.

[0160] Transposition reagents, including transposons and transposases, can be added to the nucleofection reaction of this disclosure before, simultaneously with, or after the addition of cells to the nucleofection buffer (optionally contained within the nucleofection reaction vial or cuvette). The transposons of this disclosure may include plasmid DNA, linearized plasmid DNA, PCR products, DOGGYBONE® DNA, mRNA templates, single-stranded or double-stranded DNA, protein-nucleic acid combinations, or any combination thereof. The transposons of this disclosure may include one or more sequences encoding one or more TTAA sites, one or more reverse-end repeats (ITRs), one or more long-end repeats (LTRs), one or more insulators, one or more promoters, one or more full-length or cleaved genes, one or more poly(A) signals, one or more self-cleaved 2A peptide cleavage sites, one or more intrasequence ribosome entry sites (IRESs), one or more enhancers, one or more regulators, one or more origins of replication, and any combination thereof.

[0161] The transposons of this disclosure may comprise one or more sequences encoding one or more full-length or truncated genes. The full-length and / or truncated genes introduced by the transposons of this disclosure may encode one or more of the following: signal peptides, centinrin, single-chain variable fragments (scFv), hinges, transmembrane domains, costimulatory domains, chimeric antigen receptors (CARs), chimeric T cell receptors (CAR-Ts), CARTyrin (CAR-Ts containing centinrin), receptors, ligands, cytokines, drug resistance genes, tumor antigens, allogeneic or autoantigens, enzymes, proteins, peptides, polypeptides, fluorescent proteins, mutains, or any combination thereof.

[0162] The transposons of this disclosure may be prepared with water, TAE, TBE, PBS, HBSS, culture medium, the auxiliary factors of this disclosure, or any combination thereof.

[0163] The transposons of this disclosure may be designed to optimize clinical safety and / or improve manufacturability. In a non-limiting example, the transposons of this disclosure may be designed to optimize clinical safety and / or improve manufacturability by removing unnecessary sequences or regions and / or including non-antibiotic selection markers. The transposons of this disclosure may be GMP grade or not.

[0164] The transposase enzymes of this disclosure may be encoded by sequences of one or more of plasmid DNA, mRNA, proteins, protein-nucleic acid combinations, or any combination thereof.

[0165] The transposase enzymes of this disclosure may be prepared with water, TAE, TBE, PBS, HBSS, culture medium, the auxiliary factors of this disclosure, or any combination thereof. The transposase enzymes of this disclosure, or the sequences / constructs that code or deliver, may be GMP grade or not.

[0166] The transposons and transposase enzymes of this disclosure can be delivered to cells by any means.

[0167] The compositions and methods of the present disclosure include the delivery of transposons and / or transposases to cells by plasmid DNA (pDNA), wherein the use of plasmids for delivery allows for the integration of transposons and / or transposases into the cellular chromosomal DNA, resulting in continuous transposase expression. Therefore, the transposons and / or transposase enzymes of the present disclosure may be delivered to cells as either mRNA or protein to eliminate the possibility of chromosomal integration.

[0168] The transposons and transposases of this disclosure may be pre-incubated individually or in combination with each other before introducing the transposons and / or transposases into a nucleofection reaction. The absolute amounts of each transposon and transposase, as well as their relative amounts, for example, the transposon-to-transposase ratio, can be optimized.

[0169] Optionally, following the preparation of the nucleofection reaction in a vial or cuvette, the reaction may be loaded into a nucleofector device and activated for the delivery of electrical pulses according to the manufacturer's protocol. The electrical pulse conditions used for the delivery of the transposons and / or transposases (or sequences encoding the transposons and / or transposases) of the present disclosure to cells may be optimized to produce cells with enhanced viability, higher nucleofection efficiency, better viability after nucleofection, a desired cell phenotype, and / or better / faster growth when growth techniques are added. When using Amaxa nucleofector technology, each of the various nucleofection programs of the Amaxa 2B or 4D nucleofector is intended.

[0170] Following the nucleofection reaction of this disclosure, cells can be gently added to a cell medium. For example, when T cells undergo a nucleofection reaction, T cells can be added to a T cell medium. The post-nucleofection cell medium of this disclosure may comprise one or more commercially available media. The post-nucleofection cell medium of this disclosure (including the post-nucleofection T cell medium of this disclosure) can be optimized to produce cells that exhibit better viability, higher nucleofection efficiency, better post-nucleofection viability, a more desirable cell phenotype, and / or better / faster growth when growth techniques are added. The post-nucleofection cell media of this disclosure (including the post-nucleofection T cell media of this disclosure) may include PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC medium, CTS OpTimizer T cell augmentation SFM, TexMACS medium, PRIME-XV T cell augmentation medium, ImmunoCult-XF T cell augmentation medium, and any combination thereof. The post-nucleofection cell media of this disclosure (including the post-nucleofection T cell media of this disclosure) may include one or more co-factors of this disclosure to enhance viability, nucleofection efficiency, post-nucleofection viability, cell phenotype, and / or growth with the addition of augmentation techniques. Exemplary co-factors include, but are not limited to, recombinant human cytokines, chemokines, interleukins, and any combination thereof.Exemplary cytokines, chemokines, and interleukins include IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANKL, and any combination thereof are included but not limited to these. Exemplary co-factors include but are not limited to salts, inorganic substances, metabolites, or any combination thereof. Exemplary salts, inorganics, and metabolites include, but are not limited to, HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum substitutes, antibiotics, pH adjusters, Earl's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCl, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof.Exemplary co-enhancing factors include, but are not limited to, media such as PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC medium, CTS OpTimizer T cell growth medium SFM, TexMACS medium, PRIME-XV T cell growth medium, ImmunoCult-XF T cell growth medium, and any combination thereof. Exemplary co-enhancing factors include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signaling, apoptotic pathways, and combinations thereof. Exemplary inhibitors include, but are not limited to, inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, and any combination thereof. Exemplary auxiliary factors include, but are not limited to, reagents that modify or stabilize one or more nucleic acids to enhance cell delivery, enhance nuclear delivery or transport, enhance assisted transport of nucleic acids to the nucleus, enhance the degradation of nucleic acids on chromosomes, and / or reduce DNA-mediated toxicity. Exemplary reagents that modify or stabilize one or more nucleic acids include, but are not limited to, pH adjusters, DNA-binding proteins, lipids, phospholipids, CaPO4, net-neutral charge DNA-binding peptides with or without NLS sequences, TREX1 enzymes, and any combination thereof.

[0171] The post-nucleofection cell media of the present disclosure (including the post-nucleofection T cell medium of the present disclosure) are used at room temperature or can be preheated to, for example, between 32°C and 37°C, including the endpoint. The post-nucleofection cell media of the present disclosure (including the post-nucleofection T cell medium of the present disclosure) can be preheated to any temperature that maintains or enhances cell viability and / or the expression of the transposons or parts thereof of the present disclosure.

[0172] The post-nucleofection cell cultures of this disclosure (including the post-nucleofection T cell cultures of this disclosure) may be contained in tissue culture flasks or dishes, G-Rex flasks, bioreactors or cell culture bags, or any other standard containers. The post-nucleofection cell cultures of this disclosure (including the post-nucleofection T cell cultures of this disclosure) may be left standing or, alternatively, perturbed (e.g., shaken, swirled, or vibrated).

[0173] Cell cultures after nucleofection may contain genetically modified cells. T cell cultures after nucleofection may contain genetically modified T cells. The genetically modified cells of this disclosure may be quiescent for a specified period or stimulated to grow by, for example, the addition of T Cell Expander technology. In certain embodiments, the genetically modified cells of this disclosure may be quiescent for a specified period or immediately stimulated to grow by, for example, the addition of T Cell Expander technology. The genetically modified cells of this disclosure may be quiescent for a sufficient time to adapt, sufficient time for transposition to occur, and / or sufficient time for positive or negative selection, resulting in cells with enhanced viability, higher nucleofection efficiency, better viability after nucleofection, a desirable cell phenotype, and / or better / faster growth upon the addition of expansion technology. The genetically modified cells of this disclosure may be rested for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or longer. In a particular embodiment, the genetically modified cells of this disclosure may be rested for, for example, overnight. In a particular embodiment, overnight is about 12 hours. The genetically modified cells of this disclosure may be rested for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or longer.

[0174] The genetically modified cells described herein can be selected after the nucleofection reaction and before the addition of expander technology. For optimal selection of genetically modified cells, the cells are rested in cell culture medium for at least 2–14 days after nucleofection to facilitate the identification of modified cells (e.g., differentiation of modified cells from unmodified cells).

[0175] If nucleofection of the transposons disclosed herein is successful, the expression of CAR / CARTyrin and the selection markers disclosed herein may be detectable in modified T cells as early as 24 hours post-nucleofection. Due to the chromosomal expression of the transposons, the expression of the selection markers alone cannot distinguish modified T cells (cells in which transposon integration was successful) from unmodified T cells (cells in which transposon integration was unsuccessful). When chromosomal expression of the transposons obscures the detection of modified cells by the selection markers, nucleofected cells (both modified and unmodified cells) can be quiesced for a period (e.g., 2–14 days), ceasing cell expression or losing all chromosomal transposon expression. Following this extended quiescence period, only modified T cells should remain positive for the expression of the selection markers. The length of this extended quiescence period can be optimized for each nucleofection reaction and selection process. When the expression of transposons on chromosomes obscures the detection of modified cells by selection markers, selection may be performed without this extended resting period, but additional selection steps may be included at a later point in time (e.g., either during or after the growth stage).

[0176] The selection of genetically modified cells according to this disclosure may be carried out by any means. In certain embodiments of the methods of this disclosure, the selection of genetically modified cells according to this disclosure may be carried out by isolating cells that express a specific selection marker. The selection markers of this disclosure may be encoded by one or more sequences within a transposon. The selection markers of this disclosure may be expressed by modified cells as a result of successful transposition (i.e., not encoded by one or more sequences within a transposon). In certain embodiments, the genetically modified cells according to this disclosure contain a selection marker that confers resistance to harmful compounds in the cell medium after nucleofection. Harmful compounds may include, for example, antibiotics or drugs that do not confer resistance to the modified cells by the selection marker and result in cell death. Exemplary selection markers include, but are not limited to, one or more wild-type (WT) or mutant genes: neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, and NKX2.2. Exemplary selection markers include, but are not limited to, surface-expressed selection markers or surface-expressed tags, which can be targeted by Ab-coated magnetic bead technology or column selection, respectively. Cleavable tags, such as those used in protein purification, can be added to the selection markers of the Disclosure for efficient column selection, washing, and elution. In certain embodiments, the selection markers of the Disclosure are not spontaneously expressed by modified cells (including modified T cells) and may therefore be useful in the physical isolation of modified cells (e.g., by cell sorting technology). Exemplary selection markers of the Disclosure include, but are not limited to, full-length, mutant, or truncated forms of CD271, CD19, CD52, CD34, RQR8, CD22, CD20, CD33, and any combination thereof, which are not spontaneously expressed by modified cells (including modified T cells).

[0177] The genetically modified cells of this disclosure can be selectively enlarged after a nucleofection reaction. In certain embodiments, modified T cells containing CAR / CARTyrin can be selectively enlarged by CAR / CARTyrin stimulation. Modified T cells containing CAR / CARTyrin can be stimulated by contact with a target-coated reagent (e.g., a tumor or normal cell line expressing the target, or expander beads coated with the target). Alternatively, modified T cells containing CAR / CARTyrin can be stimulated by contact with irradiated tumor cells, irradiated allogeneic normal cells, or irradiated autologous PBMCs. To minimize contamination of the cell product composition of this disclosure by the target-expressing cells used for stimulation, stimulation can be carried out using expander beads coated with the CAR / CARTyrin target protein, for example, when the cell product composition can be directly administered to the target. Selective enlargement of modified T cells containing CAR / CARTyrin by CAR / CARTyrin stimulation can be optimized to avoid functional depletion of the modified T cells.

[0178] Selected genetically modified cells of this disclosure may be cryopreserved, quized for a specified period, or stimulated for growth by adding Cell Expander technology. Selected genetically modified cells of this disclosure may be cryopreserved, quized for a specified period, or stimulated immediately for growth by adding Cell Expander technology. When the selected genetically modified cells are T cells, the T cells may be stimulated for growth by adding T-Cell Expander technology. Selected genetically modified cells of this disclosure may be quized for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or longer. In a particular embodiment, selected genetically modified cells of this disclosure may be quized overnight, for example. In a particular embodiment, overnight is about 12 hours. The selected genetically modified cells of this disclosure may be quiescent for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or longer. The selected genetically modified cells of this disclosure may be quiescent for any period of time that results in cells having enhanced viability, higher nucleofection efficiency, better viability after nucleofection, a desired cell phenotype, and / or better / faster growth when growth techniques are added.

[0179] Selected genetically modified cells (including selected genetically modified T cells of this disclosure) can be cryopreserved using any standard cryopreservation method that can be optimized for storing and / or restoring human cells with high recovery, viability, phenotype, and / or functional capacity. The cryopreservation methods of this disclosure may include commercially available cryopreservation media and / or protocols.

[0180] The transposition efficiency of selected genetically modified cells (including selected genetically modified T cells of this disclosure) can be evaluated by any means. For example, before applying expander technology, transposon expression by selected genetically modified cells (including selected genetically modified T cells of this disclosure) can be measured by fluorescence-activated cell sorting (FACS). Determining the transposition efficiency of selected genetically modified cells (including selected genetically modified T cells of this disclosure) may include determining the percentage of selected cells expressing transposons (e.g., CARs). Alternatively, or in addition, the purity of T cells, the mean fluorescence intensity (MFI) of transposon expression (e.g., CAR expression), the ability of CARs (delivered by transposons) to mediate degranulation and / or death of target cells expressing CAR ligands, and / or the phenotype of selected genetically modified cells (including selected genetically modified T cells of this disclosure) can be evaluated by any means.

[0181] The cell product compositions of this disclosure may be released for administration to a subject when certain release criteria are met. Exemplary release criteria may include, but are not limited to, certain percentages of modified, selected, and / or enlarged T cells expressing detectable levels of CARs on their cell surface.

[0182] Genetic modification of autologous T cell product composition The genetically modified cells (including genetically modified T cells) of this disclosure can be enlarged using expander technology. The expander technology of this disclosure may include commercially available expander technology. An exemplary expander technology of this disclosure involves stimulating the genetically modified T cells of this disclosure via the TCR. While all means for stimulating the genetically modified T cells of this disclosure are contemplated, stimulating the genetically modified T cells of this disclosure via the TCR is a preferred method and yields a product with a superior level of killing ability.

[0183] Thermo Expander DynaBeads can be used in a 3:1 bead-to-T cell ratio to stimulate the genetically modified T cells of this disclosure via the TCR. If the expander beads are not biodegradable, the beads may be removed from the expander composition. For example, the beads may be removed from the expander composition after about 5 days. Miltenyi T cell activation / enhancement reagents may be used to stimulate the genetically modified T cells of this disclosure via the TCR. StemCell Technologies' ImmunoCult human CD3 / CD28 or CD3 / CD28 / CD2 T cell activator reagents may be used to stimulate the genetically modified T cells of this disclosure via the TCR. This technique may be preferred because the soluble tetrameric antibody complex is degraded after a period of time and does not require removal from the process.

[0184] Artificial antigen-presenting cells (APCs) may be engineered to co-express a target antigen and used to stimulate the cells or T cells of the Disclosure by the TCR and / or CAR of the Disclosure. Artificial APCs may include or be derived from tumor cell lines (e.g., immortalized myeloid leukemia cell line K562) and may be engineered to co-express multiple costimulatory molecules or technologies (e.g., CD28, 4-1BBL, CD64, mbIL-21, mbIL-15, CAR target molecules). When the artificial APCs of the Disclosure are combined with costimulatory molecules, conditions may be optimized to prevent undesirable phenotypes and functional capabilities, i.e., the development or emergence of terminally differentiated effector T cells.

[0185] Irradiated PBMCs (autologous or allogeneic) may express several target antigens, such as CD19, and can be used to stimulate the cells or T cells of the Disclosure by the TCR and / or CAR of the Disclosure. Alternatively, or in addition, irradiated tumor cells may express several target antigens and can be used to stimulate the cells or T cells of the Disclosure by the TCR and / or CAR of the Disclosure.

[0186] Plate-bound and / or soluble anti-CD3, anti-CD2, and / or anti-CD28 stimuli can be used to stimulate the cells or T cells of the Disclosure by the TCR and / or CAR of the Disclosure.

[0187] Antigen-coated beads can present target proteins and can be used to stimulate cells or T cells of the Disclosure by the TCR and / or CAR of the Disclosure. Alternatively, or in addition, expander beads coated with CAR / CARTyrin target proteins can be used to stimulate cells or T cells of the Disclosure by the TCR and / or CAR of the Disclosure.

[0188] The amplification method involves stimulating the cells or T cells of this disclosure via TCR or CAR / CARTyrin, and via CD2, CD3, CD28, 4-1BB, and / or other markers expressed on the surface of genetically modified T cells.

[0189] The expansion technique can be applied to the cells of this disclosure from immediately after nucleofection to approximately 24 hours after nucleofection. While various cell media can be used during the expansion procedure, the preferred T cell expansion medium of this disclosure can produce cells with, for example, better viability, cell phenotype, overall expansion, or better ability to persist, engraft, and / or CAR-mediated death in vivo. The cell media of this disclosure can be optimized to improve / enhance the expansion, phenotype, and function of the genetically modified cells of this disclosure. Preferred phenotypes of expanded T cells may include a mixture of T stem cell memory, T central, and T effector memory cells. Expander Dynabeads can primarily produce central memory T cells, which can yield clinically superior performance.

[0190] The exemplary T cell growth media of this disclosure may, in part or in whole, comprise PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC medium, CTS OpTimizer T cell growth SFM, TexMACS medium, PRIME-XV T cell growth medium, ImmunoCult-XF T cell growth medium, or any combination thereof. The T cell growth media of this disclosure may further comprise one or more co-factors. The co-factors that may be included in the T cell growth media of this disclosure enhance viability, cell phenotype, overall growth, or increase in vivo persistence, engraftment, and / or CAR-mediated killing capacity. Co-factors that may be included in the T cell growth medium of this disclosure include recombinant human cytokines, chemokines, and / or interleukins, e.g., IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 This includes, but is not limited to, p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L, or any combination thereof.The co-factors that may be included in the T cell growth medium of this disclosure include, but are not limited to, salts, inorganic substances, and / or metabolites, such as HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum substitutes, antibiotics, pH adjusters, Earl's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCl, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The co-factors that may be included in the T cell growth medium of this disclosure include, but are not limited to, inhibitors of cellular DNA sensing, metabolism, differentiation, signaling, and / or apoptotic pathways, such as TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.

[0191] The auxiliary factors that may be included in the T cell growth medium of this disclosure include, but are not limited to, reagents that modify or stabilize nucleic acids to enhance cell delivery, enhance nuclear delivery or transport, enhance assisted transport of nucleic acids to the nucleus, enhance the degradation of nucleic acids on chromosomes, and / or reduce DNA-mediated toxicity, such as pH adjusters, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral-charged DNA-binding peptides having or not having an NLS sequence, TREX1 enzyme, or any combination thereof.

[0192] The genetically modified cells of this disclosure can be selected during the growth process by the use of selectable drugs or compounds. For example, in a particular embodiment, when the transposon of this disclosure can encode a selection marker that confers resistance to a drug added to the culture medium to the genetically modified cells, the selection may occur during the growth process and may require approximately 1 to 14 days of culture for the selection to occur. Examples of drug resistance genes that can be used as selection markers encoded by the transposon of this disclosure include, but are not limited to, the genes neo, DHFR, TYMS, ALDH, MDR1, MGMT, FANCF, RAD51C, GCS, NKX2.2, or any combination thereof in wild-type (WT) or mutant form.Examples of corresponding drugs or compounds that can be added to culture media and confer resistance to a selection marker include G418, puromycin, ampicillin, kanamycin, methotrexate, mephalan, temozolomide, vincristine, etoposide, doxorubicin, bendamustine, fludarabine, Aredia (disodium pamidronate), Becenum (carmustine), BiCNU ( Carmustine), Bortezomib, Carfilzomib, Carmubris (carmustine), Carmustine, Clafen (cyclophosphamide), Cyclophosphamide, Cytoxane (cyclophosphamide), Daratumumab, Darzalex (daratumumab), Doxil (doxorubicin hydrochloride liposome), Doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), E Lotuzumab, Empliciti (Elotuzumab), Evacet (Doxorubicin Hydrochloride Liposome), Farydac (Panobinostat), Ixazomib Citrate, Kyprolis (Carfilzomib), Lenalidomide, LipoDox (Doxorubicin Hydrochloride Liposome), Mozovir (Prelixafor), Neosar (Cyclophosphamide), Ninlaro (Ixazomib Citrate) This includes, but is not limited to, esters, disodium pamidronate, panobinostat, prelixafor, pomalidomide, pomalist (pomaridomide), revlimide (lenalidomide), synovir (thalidomide), thalidomide, salomid (thalidomide), velcade (bortezomib), zoledronic acid, zometa (zoledronic acid), or any combination thereof.

[0193] The T cell proliferation process described herein may occur in a cell culture bag within a WAVE bioreactor, a G-Rex flask, or any other suitable container and / or reactor.

[0194] The cells or T cell cultures of this disclosure may be stably maintained, shaken, swirled, or vibrated.

[0195] The cell or T cell augmentation processes of this disclosure can be optimized for certain conditions, including, but not limited to, the culture period, cell concentration, planned addition / removal of T cell medium, cell size, total cell number, cell phenotype, purity of cell population, percentage of genetically modified cells in the growing cell population, use and composition of supplements, addition / removal of expander technology, or any combination thereof.

[0196] The cell or T cell augmentation process of this disclosure may be continued until a predetermined endpoint before the formulation of the resulting enlarged cell population. For example, the cell or T cell augmentation process of this disclosure may be continued for a predetermined time: at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks; at least 1, 2, 3, 4, 5, 6 months, or at least 1 year. The cell or T cell augmentation process of the Disclosure may be continued until the resulting culture reaches a predetermined overall cell density: 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010 cells per volume (μl, ml, L) or any density in between. The cell or T cell augmentation process of the Disclosure may be continued until the genetically modified cells in the resulting culture exhibit a predetermined level of transposon expression of the Disclosure: 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any percentage in between (the minimum, maximum, or average level of expression indicates that the resulting modified cells are clinically effective). The cell or T cell enlargement process of this disclosure may be continued until the ratio of genetically modified cells in the resulting culture to the ratio of unmodified cells reaches a predetermined threshold: at least 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any ratio in between.

[0197] Analysis of genetically modified autologous T cells for release The percentage of genetically modified cells can be assessed during or after the growth process of the Disclosure. Transposon expression by the genetically modified cells of the Disclosure can be measured by fluorescence-activated cell sorting (FACS). For example, FACS can be used to determine the percentage of cells or T cells expressing the CARs of the Disclosure. Alternatively, or in addition, the purity of the genetically modified cells or T cells, the mean fluorescence intensity (MFI) of the CARs expressed by the genetically modified cells or T cells of the Disclosure, the ability of the CARs to mediate degranulation and / or death of target cells expressing the CAR ligands, and / or the phenotype of CAR+ T cells can be assessed.

[0198] Compositions of the Disclosure intended for administration to a subject may need to meet one or more “release criteria” indicating that the composition is safe and effective as a pharmaceutical formulation and / or for administration to a subject. Release criteria may include the requirement that the composition of the Disclosure (e.g., the T cell product of the Disclosure) contains a certain percentage of T cells expressing a detectable level of the CAR of the Disclosure on their cell surface.

[0199] The growth process should continue until certain criteria are met (e.g., achieving a specific total number of cells, achieving a specific population of memory cells, achieving a population of a specific size).

[0200] Certain criteria signal when the growth process should end. For example, cells should be formulated, reactivated, or cryopreserved when their cell size reaches 300 fL (otherwise, cells that reach a size beyond this threshold may begin to die). Cryopreservation immediately after the cell population reaches an average cell size of less than 300 fL may result in better cell recovery during thawing and culture because the cells have not yet reached a completely quiescent state before cryopreservation (the complete quiescent size is approximately 180 fL). Before proliferation, the T cells of this disclosure may have a cell size of approximately 180 fL, but three days after proliferation, their cell size may increase more than fourfold to approximately 900 fL. Over the next 6–12 days, the T cell population slowly decreases in cell size until it is completely quiescent at 180 fL.

[0201] The process for preparing a cell population for a formulation may include, but is not limited to, steps of enriching the cells of the cell population, washing the cells, and / or further selection of cells via sorting magnetic beads for drug resistance or specific surface expression markers. The process for preparing a cell population for a formulation may further include a sorting step to ensure the safety and purity of the final product. For example, if tumor cells from a patient have been genetically modified to stimulate the genetically modified T cells of this disclosure, or to stimulate the genetically modified T cells of this disclosure that are being prepared for a formulation, it is important that tumor cells from the patient are not included in the final product.

[0202] Cell product injection and / or cryopreservation for injection The pharmaceutical formulations of this disclosure may be dispensed into bags for infusion, cryopreservation, and / or storage.

[0203] The pharmaceutical formulations of the present disclosure can be cryopreserved using standard protocols and optionally an injectable cryopreservation medium. For example, cryopreservatives without DMSO (e.g., CryoSOfree™ DMSO-free cryopreservation medium) can be used to reduce the toxicity associated with freezing. The cryopreserved pharmaceutical formulations of the present disclosure can be stored for later administration to patients. Effective treatment may require multiple administrations of the pharmaceutical formulations of the present disclosure, and thus the pharmaceutical formulations can be stored frozen but packaged in pre-allocated "doses" that are separated for thawing of individual doses.

[0204] The pharmaceutical formulations of the present disclosure may be stored at room temperature. Effective treatment may require multiple administrations of the pharmaceutical formulations of the present disclosure, and thus the pharmaceutical formulations can be stored together but packaged in pre-allocated "doses" that are separated for administration of individual doses.

[0205] The pharmaceutical formulations of the present disclosure can be recorded for subsequent re-proliferation and / or selection for the generation of further doses for the same patient who may need to be administered in the future, for example, in the case of allogeneic therapy after remission and recurrence of the condition.

[0206] Formulation As described above, this disclosure provides stable formulations, preferably comprising phosphate buffer and saline or a selected salt, containing at least one protein scaffold in a pharmaceutically acceptable formulation, as well as preservative solutions and formulations containing preservatives, and multiple-use preservative formulations suitable for pharmaceutical or veterinary use. The preservative formulations contain at least one known preservative, or optionally, at least one selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercury nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, polymers, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture may be used, as is known in the art, such as about 0.0015%, or any range, value, or fractions between them. Non-limiting examples include no preservatives, approximately 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), approximately 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), approximately 0.001-0.5% thimerosal (e.g., 0.005, 0.01%), and approximately 0.001-2.0% phenol (e.g., This includes 0.05%, 0.25%, 0.28%, 0.5%, 0.9%, 1.0%, 0.0005-1.0% alkylparabens (e.g., 0.00075%, 0.0009%, 0.001%, 0.002%, 0.005%, 0.0075%, 0.009%, 0.01%, 0.02%, 0.05%, 0.075, 0.09, 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 0.9%, 1.0%), etc.

[0207] As described above, the present invention provides a manufactured product comprising a packaging material and at least one vial containing a solution of at least one protein scaffold, optionally in an aqueous diluent, having a formulated buffering agent and / or preservative, wherein the packaging material includes a label indicating that the solution can be held for 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or longer. The present invention further includes a product comprising a packaging material, a first vial containing a lyophilized at least one protein scaffold, and a second vial containing an aqueous diluent of a predetermined buffering agent or preservative, wherein the packaging material includes a label instructing the patient to reconstitute the at least one protein scaffold with the aqueous diluent to form a solution that can be held for 24 hours or longer.

[0208] At least one protein scaffold used according to the present invention can be produced by recombinant means including obtaining from mammalian cells or transgenic preparations, or can be purified from other biological resources as described herein or known in the art.

[0209] The range of at least one protein scaffold in the products of the present invention, in the case of wet / dry systems, includes amounts that result in concentrations of from about 1.0 μg / ml to about 1000 mg / ml upon reconstitution, although lower and higher concentrations are possible and depend on the intended delivery vehicle, e.g., solution formulations are different from transdermal patches, lungs, transmucosal, or osmotic, or micropump methods.

[0210] Preferably, the aqueous diluent further optionally comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of the preservative used in the formulation is sufficient to obtain an antimicrobial effect. Such a concentration depends on the selected preservative and is readily determined by those skilled in the art.

[0211] Other excipients, such as isotonic agents, buffers, antioxidants, and preservatives, may be optionally added, preferably to the diluent. Isotonic agents, such as glycerin, are generally used at known concentrations. Physiologically tolerable buffers are preferably added to provide improved pH control. The formulations can cover a wide range of pH, such as a preferred range of about pH 4 to about pH 10, a preferred range of about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH of about 6.8 to about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, and especially phosphate-buffered saline (PBS).

[0212] Aggregation can be reduced by optionally adding pharmaceutically acceptable solubilizers such as Tween20 (polyoxyethylene(20) sorbitan monolaurate), Tween40 (polyoxyethylene(20) sorbitan monopalmitate), Tween80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol), or nonionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, as well as chelating agents such as other block copolymers like Pluronic®, EDTA, and EGTA. These additives are particularly useful when the formulation is administered using a pump or plastic container. The presence of pharmaceutically acceptable surfactants reduces the tendency of proteins to aggregate.

[0213] The formulations of the present invention can be prepared by mixing at least one protein scaffold with phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. Mixing at least one protein scaffold and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one protein scaffold in a buffered solution is combined with a desired preservative in a buffered solution in an amount suitable for providing the protein and preservative at the desired concentration. Variations in this process will be recognized by those skilled in the art. For example, the order in which components are added when additional additives are used, the temperature and pH in which the formulation is prepared are all factors that can be optimized for the concentration and means of administration used.

[0214] The requested formulation may be provided to the patient as a clear solution, or as two vials, comprising a lyophilized vial of at least one protein scaffold, which is reconstituted in a second vial containing water, a preservative, and / or an excipient, preferably a phosphate buffer, and / or saline and a selected salt in an aqueous diluent. The single-solution vial or the two vials requiring reconstitution can be reused multiple times and adequately accommodate one or more cycles of patient treatment, thus providing a more convenient treatment regimen than those currently available.

[0215] The claimed product is useful for administration over a period ranging from immediate to 24 hours or more. Therefore, the claimed product provides significant benefits to the patient. The formulation of the present invention can optionally be safely stored at temperatures of about 2°C to about 40°C and retains the biological activity of the protein for an extended period, allowing for packaging labeling indicating that the solution can be retained and / or used for 6, 12, 18, 24, 36, 48, 72, or 96 hours or longer. When using a stored dilution, such labeling may include use for up to 1 to 12 months, 6 months, 1.5 years, and / or 2 years.

[0216] A solution of at least one protein scaffold of the present invention can be prepared by a process comprising mixing at least one protein scaffold in an aqueous diluent. The mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, measured amounts of at least one protein scaffold in water or buffer are combined with the protein and, optionally, a preservative or buffer in an amount suitable for providing the desired concentration. Variations in this process will be recognized by those skilled in the art. For example, the order in which the components are added, whether additional additives are used, and the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and means of administration used.

[0217] The requested product may be provided to the patient as a clear solution or as two vials, each containing at least one lyophilized protein scaffold vial, which is reconstituted in a second vial containing an aqueous diluent. The single-solution vial or the two vials requiring reconstitution can be reused multiple times and adequately accommodate one or more cycles of patient treatment, thus providing a more convenient treatment regimen than those currently available.

[0218] The requested product may be indirectly provided to patients by providing a pharmacy, clinic, or institution and facility with two vials, each containing a vial of lyophilized at least one protein scaffold, which is reconstituted in a second vial containing a clear solution or an aqueous diluent. The clear solution in this case may be up to 1 liter or larger in size and provide a large reservoir from which smaller portions of the at least one protein scaffold solution can be taken out once or more times and transferred to smaller vials, which are provided by the pharmacy or clinic to their customers and / or patients.

[0219] Approved devices including single vial systems include pen-type syringe devices for solution delivery, such as BD Pens, BD Autojector®, Humaject®, NovoPen®, BD® Pen, AutoPen®, and OptiPen®, GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, and Medi-Ject®, manufactured or developed by companies such as Becton Dickinson (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com), Bioject, Portland, Oregon (www.bioject.com), and National Medical Products from Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn., www.mediject.com), and similar suitable devices are among those approved. Approved devices including two-vial systems include these pen-type injector systems for reconstituting lyophilized drugs in cartridges for delivery of reconstituted solutions, such as HumatroPen®. Examples of other suitable devices include pre-filled syringes, auto-injectors, needleless injectors, and needleless IV infusion sets.

[0220] The claimed product includes packaging materials. The packaging materials provide conditions for the use of the product, in addition to information required by regulatory authorities. The packaging materials of the present invention provide instructions to patients to use the solution for a period of 2 to 24 hours or more for two vial products, wet / dry, by reconstituting at least one protein scaffold in an aqueous diluent to form a solution. For single vial solution products, the label indicates that such solution is usable for 2 to 24 hours or more. The currently claimed product is useful for human medicinal use.

[0221] The formulations of the present invention can be prepared by a step comprising mixing at least one protein scaffold with a selected buffer, preferably physiological saline or a phosphate buffer containing a selected salt. Mixing at least one protein scaffold and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one protein scaffold in water or buffer is combined with a desired buffer in water in sufficient quantity to provide the protein and buffer at the desired concentration. Variations in this step will be recognized by those skilled in the art. For example, the order in which components are added, whether additional additives are used, and the temperature and pH at which the formulation is prepared are all factors that can be optimized for the concentration and means of administration used.

[0222] The requested stable or preserved formulation can be provided to the patient as a clear solution, or as a two-vial set containing a vial of lyophilized protein scaffolding which is reconstituted in a second vial containing a preservative or buffer and excipients in an aqueous diluent. The single-solution vial or the two-vial set requiring reconstitution can be reused multiple times and adequately accommodate one or more cycles of patient treatment, thus providing a more convenient treatment regimen than those currently available.

[0223] Other formulations or methods for stabilizing protein scaffolds may result in something other than a clear solution of lyophilized powder containing the protein scaffold. Among opaque solutions, formulations may include a suspension of microparticles, the microparticles being a composition containing a variable-dimensional protein scaffold, and are variously known as microspheres, microparticles, nanoparticles, nanospheres, or liposomes. Relatively homogeneous, essentially spherical particle formulations containing activators can be formed by contacting an aqueous phase containing the activator and polymer with a non-aqueous phase, followed by evaporation of the non-aqueous phase, which causes the particles to coalesce from the aqueous phase, as taught in U.S. Patent No. 4,589,330. Porous microparticles can be prepared using a first phase containing the activator and polymer dispersed in a continuous solvent, and by removing the solvent from the suspension by lyophilization or dilution-extraction-precipitation, as taught in U.S. Patent No. 4,818,542. Preferred polymers for such preparations include gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycoside-L(-)lactide, poly(epsilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(β-hydroxybutyric acid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate), polyamide, poly(amino acid), poly(2-hydroxyethyl DL-aspartamide), poly(ester urea), poly(L-phenylalanine / ethyl The polymer is a natural or synthetic copolymer or polymer selected from polyglycol / 1,6-diisocyanatohexane and poly(methyl methacrylate). Particularly preferred polymers are polyesters such as polyglycolic acid, polylactic acid, glycolide-L(-)lactide, poly(epsilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid), and poly(epsilon-caprolactone-CO-glycolic acid). Useful solvents for dissolving the polymer and / or active substance include water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate.The step of dispersing the active substance-containing phase in the second phase may include pressurizing the first phase and passing it through the nozzle opening to influence droplet formation.

[0224] Dry powder formulations may arise from processes other than freeze-drying, such as by spray-drying by evaporation, solvent extraction, or precipitation of a crystalline composition followed by one or more steps to remove an aqueous or non-aqueous solvent. The preparation of spray-dried protein scaffold preparations is taught in U.S. Patent No. 6,019,968. Protein scaffold-based dry powder compositions can be produced by spray-drying a solution or slurry of a protein scaffold and optionally an excipient in a solvent under conditions that provide an inhalable dry powder. The solvent may include polar compounds that can be easily dried, such as water and ethanol. The stability of the protein scaffold can be enhanced by performing the spray-drying procedure in the absence of oxygen, such as under a nitrogen blanket, or by using nitrogen as the drying gas. Another relatively dry formulation is a dispersion of multiple porous microstructures dispersed in a suspension medium, typically containing a hydrofluoroalkane propellant, as taught in WO9916419. Stable dispersions can be administered to the patient's lungs using a metered-dose inhaler. Equipment useful for the commercial manufacture of spray-dried pharmaceuticals is manufactured by Buchi Ltd. or Niro Corp.

[0225] At least one protein scaffold in a stable or stored formulation or solution described herein can be administered to a patient in accordance with the present invention by various delivery methods, including SC or IM injection, percutaneous, pulmonary, transmucosal, implant, osmotic pump, cartridge, micropump, or other means as well understood by those skilled in the art.

[0226] Applications in treatment The present invention also provides a method for modulating or treating a disease in cells, tissues, organs, animals, or patients, for example, by administering or contacting cells, tissues, organs, animals, or patients with a therapeutically effective amount of the protein scaffold, using at least one of the protein scaffolds of the present invention, as is known in the art or as described herein. The present invention also provides a method for modulating or treating a disease in cells, tissues, organs, animals, or patients, including but not limited to malignant diseases.

[0227] The present invention also provides a method for modulating or treating at least one cancer or malignant disease in a cell, tissue, organ, animal, or patient, including but not limited to leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma, relapsed multiple myeloma, refractory multiple myeloma, Kaposi sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, tumor associated syndrome / hypercalcemia associated with malignant disease, solid tumor, bladder cancer, breast cancer, triple negative breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, castration-resistant prostate cancer, renal cell cancer, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, etc. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is relapsed multiple myeloma. In some embodiments, the cancer is refractory multiple myeloma. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is castration-resistant prostate cancer (CRPC). In some embodiments, the solid cancer is breast cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, or renal cancer. In some embodiments, the breast cancer is triple negative breast cancer.

[0228] This disclosure provides a method for treating cancer, comprising administering to a subject a first composition comprising a population of T cells expressing a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-recognition domain, and a second composition comprising an anti-CD20 agent. In some embodiments, the anti-CD20 agent is rituximab, ofatumumab, ocrelizumab, iodine i131 tositumomab, obinutuzumab, or ibritumomab. In some embodiments, the anti-CD20 agent is rituximab (RITUXAN®).

[0229] In some embodiments, administration of CAR-T and anti-CD20 agents to subjects results in increased in vivo survival and persistence of CAR-T compared to subjects administered CAR-T alone. In some embodiments, CAR-T copies / mL or CAR-T copies / ugDNA in blood samples are determined over time as a surrogate for persistence. In some embodiments, the area under the plasma concentration curve (AUC) (i.e., the area defined by the uppermost plasma concentration curve and the lowermost x-axis (time)) is used as a measure of persistence. In some embodiments, the AUC of the plasma concentration curve after 100 days (hours) is used as a measure of persistence. Increased persistence of CAR-T in subjects provides a superior effect of improved response to treatment. Increased AUC in subjects provides a superior effect of improved response to treatment. The “increased” or “enhanced” amount is typically a “statistically significant” amount and may include an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, or 3000% (including all percentage increases between these, e.g., 11%, 12%, 13%, 14%, 15%) greater than the response in subjects treated with CAR alone. The “increased” or “enhanced” amounts may include increases of 1%–10%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, 90%–100%, 100%–150%, or 150%–200% greater than the response of subjects treated with CAR-T alone. In some embodiments, the persistence of CAR-T in subjects treated with CAR-T and anti-CD20 agents is at least 75% higher compared to subjects treated with CAR-T alone. In some embodiments, the persistence of CAR-T in subjects treated with CAR-T and anti-CD20 agents is at least 90% higher compared to subjects treated with CAR-T alone.

[0230] In some embodiments, administration of CAR and anti-CD20 agents to subjects results in increased in vivo persistence and expansion of CAR in subjects compared to subjects administered CAR alone. In some embodiments, administration of CAR and anti-CD20 agents to subjects results in increased in vivo persistence but does not affect CAR expansion in subjects compared to subjects administered CAR alone.

[0231] As used herein, the terms “anti-drug antibody” or “ADA” refer to antibodies produced in a subject against a therapeutic protein present in that subject. Classical anti-drug antibody (ADA) responses are understood in the art to result from systemic administration of recombinant therapeutic proteins to a subject. Furthermore, as used herein in relation to CAR-T therapeutics, the ADA response is intended to encompass antibody responses observed in the studies described herein, which produce antibodies that bind to CAR-T (i.e., antibodies produced against P-BCMA-101 CAR-T).

[0232] In some embodiments, administration of CAR-T and anti-CD20 agents to subjects results in a reduced anti-drug antibody (ADA) response to CAR-T compared to subjects administered CAR-T alone. In some embodiments, the ADA response is measured over time in peripheral blood using a meso-scale discovery (MSD) assay. The reduction in the ADA response in subjects provides a superior effect of an improved response to treatment. The amount of “reduced” or “downgraded” is typically a “statistically significant” amount and may include reductions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (including all increasing percentages between, e.g., 11%, 12%, 13%, 14%, 15%) lower than the response in subjects administered CAR-T alone. The “reduced” or “downgraded” amounts may include reductions of 1%–10%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% lower than the response in subjects administered with CAR-T alone. In some embodiments, the anti-drug antibody response to CAR-T in subjects administered with CAR-T and anti-CD20 agents was at least 50% lower compared to subjects administered with CAR-T alone.

[0233] Any method of the present invention may include administering an effective amount of a composition or pharmaceutical composition comprising at least one protein scaffold to cells, tissues, organs, animals, or patients requiring such regulation, treatment, or therapy. Such methods may optionally further include co-administration or combination therapy for treating such disease or disorder, further comprising administering the at least one protein scaffold, a particular portion thereof, or a variant before, simultaneously with, and / or after, at least one selected from at least one alkylating agent, mitotic inhibitor, and radiopharmaceutical. Preferred dosages are well known in the art. For example, see Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000), PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001, Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ, each of these references is incorporated herein by reference in whole.

[0234] Preferred doses may be optionally comprised of approximately 0.1 to 99 and / or 100 to 500 mg / kg / administered dose, or any range, value, or fraction thereof, or one or more doses, or any range, value, or fraction thereof, to achieve serum concentrations of approximately 0.1 to 5000 μg / ml. Preferred dose ranges for the protein scaffold of the present invention are approximately 1 mg to a maximum of approximately 3, approximately 6, or approximately 12 mg / kg of patient body weight.

[0235] Alternatively, the dosage administered can vary depending on known factors such as the pharmacodynamic properties of the specific drug, its mode and route of administration, the recipient's age, health condition, and weight, the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Typically, the dosage of the active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight. Usually, 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram of body weight per dose or in sustained-release form is effective in obtaining the desired results.

[0236] As a non-limiting example, treatment for humans or animals may be provided using single injections or repeated doses, with at least one protein scaffold of the present invention as a single or periodic dose, approximately 0.1 to 100 mg / kg per day or any range, value, or fraction thereof, at least once within 1 to 40 days, or alternatively or additionally, at least once within 1 to 52 weeks, or alternatively or additionally, at least once within 1 to 20 years, or any combination thereof.

[0237] Suitable dosage forms (compositions) for internal administration generally contain approximately 0.001 milligrams to approximately 500 milligrams of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is typically present in an amount of approximately 0.5 to 99.999% by weight based on the total weight of the composition.

[0238] In some embodiments, the first composition, comprising a population of T cells expressing P-BCMA-101 CAR, contains at least 0.1 × 10⁶ cells. 6 , 0.2 × 10 6 , 0.25 × 10 6 , 0.5 × 10 6 , 0.6 × 10 6 , 0.7 × 10 6 , 0.75 × 10 6 , 0.8 × 10 6 , 0.9 × 10 6 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×106 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 10×10 6 , 11×10 6 , 12×10 6 , 13×10 6 , 14×10 6 , 15×10 6 , 16×10 6 , 17×10 6 , 18×10 6 , 19×10 6 , or 20×10 6 are administered at a total dosage of the target body weight of cells / kg. In some embodiments, the CAR-T cells are administered at a dosage of cells / kg / dose. In some embodiments, the CAR-T cells are administered at a dosage of cells / kg / dose. In some embodiments, the CAR-T cells are administered at a dosage of cells / kg / dose. In some embodiments, the CAR-T cells are administered at a dosage of cells / kg / dose. In some embodiments, the CAR-T cells are administered at a dosage of cells / kg / dose. In some embodiments, the CAR-T cells are administered at a dosage of cells / kg / dose.

[0239] ​​​​​​​​​​​​For parenteral administration, the protein scaffold can be formulated as a solution, suspension, emulsion, particles, powder, or lyophilized powder, either in association with or separately provided with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and about 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixative oils can also be used. The vehicle or lyophilized powder may contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulations are sterilized by known or preferred techniques.

[0240] Suitable pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference in this field.

[0241] Alternative administration Many known and developed methods can be used in accordance with the present invention to administer a pharmaceutically effective amount of at least one protein scaffold according to the present invention. Although pulmonary administration is used in the following description, other administration methods can be used in accordance with the present invention with suitable results. The protein scaffold of the present invention can be delivered in a carrier as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or by other methods described herein or known in the art.

[0242] Parenteral formulations and administration Preparations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogenated naphthalene, etc. Aqueous or oily suspensions for injection may be prepared according to known methods using appropriate emulsifiers or wetting agents and suspending agents. Drugs for injection may be non-toxic, parenterally administered diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Acceptable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile non-volatile oils may be used as ordinary solvents or suspension solvents. For these purposes, all kinds of non-volatile oils and fatty acids may be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids, and natural, synthetic, or semi-synthetic mono-, di-, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection methods, gas-pressurized needleless injection devices described in U.S. Patent No. 5,851,198, and laser puncture devices described in U.S. Patent No. 5,839,446, which are incorporated herein by reference in their entirety.

[0243] alternative delivery The present invention further relates to the delivery of at least one protein scaffold by non-oral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracolonic, intracerebral, intracerebral, intracerebral, intracerebral, intracerebral, intracerebral, intracardiac, intracardiac, intramuscular, intrastellar, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intraneural, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intrabladder, intrafocal, bolus, vagina, rectum, cheek, sublingual, intranasal, or percutaneous means. At least one protein scaffold composition may be used parenterally (subcutaneous, intramuscular, or intravenous) or for any other administration, particularly in the form of a solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms, such as creams and suppositories, but not limited to these; for cheek or sublingual administration, in forms such as tablets or capsules, but not limited to these; for intranasal administration, in the form of powders, nasal drops or aerosols, or certain drugs, but not limited to these; or transdermally, in the form of gels, ointments, lotions, suspensions, or having a chemical enhancer such as dimethyl sulfoxide to either improve skin structure or increase drug concentration in a transdermal patch (Junginger, et al. "Drug Permeation Enhancement" Hsieh, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New It can be prepared for use such as, but not limited to, patch delivery systems having an oxidizing agent that enables the application of formulations containing proteins and peptides in the skin (WO98 / 53847), or for the application of an electric field to create a temporary transport pathway, such as electroporation, or for increasing the mobility of charged drugs through the skin, such as iontophoresis, or for the application of ultrasound, such as sonophoresis (U.S. Patents No. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0244] Lung / nasal administration For pulmonary administration, preferably, at least one protein scaffold composition is delivered in a particle size effective for reaching the lungs or the lower respiratory tract of the sinuses. According to the present invention, at least one protein scaffold can be delivered by any of the various inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations into the patient's sinuses or alveoli, include metering inhalers, nebulizers, dry powder generators, and nebulizers. Other devices suitable for managing pulmonary or nasal administration of protein scaffolds are also known in the art. All such devices can use formulations suitable for administration to distribute the protein scaffold in an aerosol. Such aerosols can consist of either a solution (both aqueous and non-aqueous) or solid particles.

[0245] Measuring-dose inhalers, such as the Ventolin Measuring-Dose Inhaler, typically use a propellant gas and require activation during inhalation (see, e.g., WO94 / 16970, WO98 / 35888). Dry powder inhalers, such as Turbuhaler® (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros® Inhalers (Dura), devices marketed by Inhale Therapeutics, and Spinhaler® Powder Inhalers (Fisons), use respiratory activation of a mixed powder (U.S. Patent No. 4,668,218 Astra, EP237507 Astra, WO97 / 25086 Glaxo, WO94 / 08552 Dura, U.S. Patent No. 5,458,135 Inhale, WO94 / 06498 Fisons, the whole incorporated herein by reference). Nebulizers such as AERx® Aradigm, Ultravent® nebulizer (Mallinckrodt), and Acorn II® nebulizer (Marquest Medical Products) (U.S. Patent No. 5,404,871 Aradigm, WO97 / 22376), which are incorporated herein by reference in their entirety, generate aerosols from solutions, while metered-dose inhalers, dry powder inhalers, etc., generate fine-particle aerosols. These specific examples of commercially available inhalation devices are intended to be representative of specific devices suitable for carrying out the present invention and are not intended to limit the scope of the present invention.

[0246] Preferably, the composition comprising at least one protein scaffold is delivered by a dry powder inhaler or nebulizer. The inhalation device for administering at least one protein scaffold of the present invention has several desirable features. For example, delivery by the inhalation device is reliable, reproducible, and accurate as advantages. The inhalation device can optionally deliver dry small particles, e.g., less than about 10 μm, preferably about 1 to 5 μm, for excellent breathability.

[0247] Administration of protein scaffold composition as a spray A spray containing a protein scaffold composition can be produced by passing a suspension or solution of at least one protein scaffold through a nozzle under pressure. The desired output and particle size can be achieved by selecting the nozzle size and configuration, the applied pressure, and the liquid supply rate. Electrospray can be produced, for example, by an electric field associated with a capillary or nozzle feed. Advantageously, the particles of the at least one protein scaffold composition delivered by the sprayer have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

[0248] A formulation of at least one protein scaffold composition suitable for use with a sprayer typically contains the protein scaffold composition in an aqueous solution at a concentration of at least one protein scaffold composition of about 0.1 mg to about 100 mg or mg / gm per ml of solution, or any range, value, or fraction thereof. The formulation may contain excipients, buffers, isotonic agents, preservatives, surfactants, and preferably agents such as zinc. The formulation may also contain excipients or agents for stabilizing the protein scaffold composition, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful for formulating protein scaffold compositions include albumin and protamine. Typical carbohydrates useful for formulating protein scaffold compositions include sucrose, mannitol, lactose, trehalose, and glucose. The protein scaffold composition formulation may also contain surfactants, which can reduce or prevent surface-induced aggregation of the protein scaffold composition caused by atomization of the solution when forming an aerosol. Various conventional surfactants can be used, including polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene sorbitol fatty acid esters. The amount is generally in the range of 0.001 to 14% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, etc. Additional agents known in the art for the formulation of proteins such as protein scaffolds, or specific parts or variants of proteins, may also be included in the formulation.

[0249] Administration of protein scaffold composition by nebulizer The protein scaffold composition of the present invention can be administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, a jet nebulizer uses a compressed air source to generate a high-speed air jet through an opening. As the gas expands beyond the nozzle, a low-pressure region is created, drawing out a solution of the protein scaffold composition through a capillary connected to a liquid reservoir. The liquid flow from the capillary is sheared into unstable filaments and droplets as it exits the tube, producing an aerosol. Various configurations, flow rates, and baffle types can be used to achieve desired performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, high-frequency electrical energy is used to generate oscillating mechanical energy, usually using a piezoelectric transducer. This energy is transferred to the protein scaffold composition formulation, either directly or via a connecting fluid, to produce an aerosol containing the protein scaffold composition. Advantageously, the particles of the protein scaffold composition delivered by the nebulizer have a particle size in the range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

[0250] A formulation of at least one protein scaffold suitable for use with either a jet or ultrasonic nebulizer typically comprises at least one protein scaffold at a concentration of about 0.1 mg to about 100 mg per ml of solution. The formulation may contain excipients, buffers, isotonic agents, preservatives, surfactants, and preferably agents such as zinc. The formulation may also contain excipients or agents for stabilizing the at least one protein scaffold composition, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful for formulating the at least one protein scaffold composition include albumin, protamine, and the like. Typical carbohydrates useful for formulating the at least one protein scaffold include sucrose, mannitol, lactose, trehalose, glucose, and the like. The at least one protein scaffold formulation may also contain surfactants that can reduce or prevent surface-induced aggregation of the at least one protein scaffold caused by atomization of the solution when forming an aerosol. A variety of conventional surfactants can be used, including polyoxyethylene fatty acid esters and alcohols, as well as polyoxyethylene sorbitol fatty acid esters. The amount is generally in the range of about 0.001 to 4% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art for the formulation of proteins, such as protein scaffolds, may also be included in the formulation.

[0251] Administration of protein scaffold composition using a metered-dose inhaler. In a medium-dose inhaler (MDI), a propellant, at least one protein scaffold, and any excipients or other additives are contained in a canister as a mixture including liquefied compressed gas. The operation of the metering valve releases the mixture as an aerosol, preferably containing particles in a size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. Desired aerosol particle sizes can be obtained by using formulations of protein scaffold compositions produced by various methods known to those skilled in the art, including jet milling, spray drying, critical point condensation, etc. Preferred MDIs include those manufactured by 3M or Glaxo and using hydrofluorocarbon propellants. Formulations of at least one protein scaffold for use with MDI devices generally comprise a suspension in a non-aqueous medium, for example, of a fine powder containing at least one protein scaffold suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, and includes trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), etc. Preferably, the propellant is a hydrofluorocarbon. A surfactant can be selected to stabilize at least one protein scaffold as a suspension in the propellant, to protect the activator from chemical decomposition, etc. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid, etc. In some cases, it is preferable to use a solvent such as ethanol for the solution aerosol. Additional agents known in the art for protein formulation can also be included in the formulation. Those skilled in the art will recognize that the method of the present invention can be achieved by pulmonary administration of at least one protein scaffold composition via a device not described herein.

[0252] Oral preparations and administration Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase intestinal wall permeability, and co-administration of enzyme inhibitors that inhibit enzymatic degradation (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFF), and trazilol). Formulations for the delivery of hydrophilic agents, comprising proteins and protein scaffolds, and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, vaginal transfemoral, or rectal administration, are taught in U.S. Patent No. 6,309,663. The active ingredient compound in solid-state dosage forms for oral administration may be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, and fragrances.

[0253] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration include emulsions, syrups, elixirs, suspensions, and solution preparations that are acceptable for medical use. These preparations may contain inert diluents commonly used in the art, such as water. Liposomes have also been described as a drug delivery system for insulin and heparin (U.S. Patent No. 4,239,754). More recently, microspheres of artificial polymers (proteinoids) of mixed amino acids have been used to deliver pharmaceuticals (U.S. Patent No. 4,925,673). Furthermore, carrier compounds used for orally delivering bioactive agents, as described in U.S. Patents No. 5,879,681 and U.S. Patent No. 5,871,753, are known in the art.

[0254] Mucosal preparations and administration Formulations for orally administering a bioactive agent encapsulated in one or more biocompatible polymers or copolymer excipients, preferably biodegradable polymers or copolymers, result in microcapsules that, due to the appropriate size of the resulting microcapsules, reach and take up the drug in aggregated lymphoid nodules (otherwise also known as "Peyer's patches" or "GALT" in animals) without loss of potency due to the drug passing through the gastrointestinal tract. Similar aggregated lymphoid nodules may also be found in the bronchi (BALT) and the large intestine. The above tissues are generally referred to as mucosa-associated lymphoid reticular tissue (MALT). Compositions and methods for administering at least one protein scaffold for absorption across the mucosal surface include an emulsion comprising a plurality of submicron particles, mucosal-adherent macromolecules, bioactive peptides, and an aqueous continuous phase, thereby promoting absorption across the mucosal surface by achieving mucosal adhesion of the emulsion particles (U.S. Patent No. 5,514,670). Suitable mucosal surfaces for application of the emulsion of the present invention may include the cornea, conjunctiva, cheek, sublingual, nose, vagina, lung, stomach, intestine, and rectum as routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycol, petrolatum, and cocoa butter. Formulations for intranasal administration may be solid and may contain excipients such as lactose or be an aqueous or oily solution of a nasal spray. For buccal administration, excipients may include sugars, calcium stearate, magnesium stearate, and pregelatinized starch (U.S. Patent No. 5,849,695).

[0255] Transdermal preparations and administration For transdermal administration, at least one protein scaffold is encapsulated in a delivery device such as liposomes or polymer nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known and include microparticles consisting of polyhydroxy acids such as polylactic acid, polyglycolic acid, and copolymers thereof, polyorthoesters, polyanhydrides, and synthetic polymers such as polyphosphazenes, as well as collagen, polyamino acids, albumin and other proteins, alginic acid and other saccharides, and combinations thereof (U.S. Patent No. 5,814,599).

[0256] Extended administration and formulations It may be desirable to deliver the compounds of the present invention over an extended period, for example, from a single dose to a period of 1 week to 1 year. Various sustained-release, depot, or implantable administration forms can be utilized. For example, the administration form may include pharmaceutically acceptable nontoxic salts of compounds having low solubility in body fluids, such as (a) acid addition salts having polybases such as phosphoric acid, sulfuric acid, citrate, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acid, or polygalacturonic acid; (b) salts having polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium, or organic cations formed from, for example, N,N'-dibenzyl ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), for example, zinc tannate salt. In addition, the compounds of the present invention, or preferably relatively insoluble salts such as those described above, can be formulated into aluminum monostearate gels, for example, using sesame oil, which are suitable for injection. Particularly preferred salts include zinc salts, zinc tannate salts, pamoate salts, etc. Another type of sustained-release depot formulation for injection contains the compounds or salts dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as polylactic acid / polyglycolic acid polymers, as described in U.S. Patent No. 3,773,919, for example. The compounds, or preferably relatively insoluble salts such as those described above, can also be formulated into cholesterol matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, JR Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0257] Injection of modified cells as adoptive cell therapy This disclosure provides modified cells expressing one or more CARs and / or CARTyrins of this disclosure, which are selected and / or enlarged for administration to subjects requiring them. The modified cells of this disclosure may be formulated for storage at any temperature, including room temperature and body temperature. The modified cells of this disclosure may be formulated for cryopreservation and subsequent thawing. The modified cells of this disclosure may be formulated in a pharmaceutically acceptable carrier for direct administration to subjects from sterile packaging. The modified cells of this disclosure may be formulated in a pharmaceutically acceptable carrier with indicators of cell viability and / or CAR / CARTyrin expression levels to ensure minimum levels of cellular function and CAR / CARTyrin expression. The modified cells of this disclosure may be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further proliferation and / or prevent cell death.

[0258] Inducible apoptosis-promoting polypeptides The inducible pro-apoptotic polypeptides of this disclosure are superior to existing inducible polypeptides because they are far less immunogenic. The inducible pro-apoptotic polypeptides of this disclosure are recombinant polypeptides and therefore do not contain non-human sequences that, if non-spontaneously generated, are recombined to produce the inducible pro-apoptotic polypeptides of this disclosure, which may be recognized as “non-self” by the host human immune system, and consequently induce an immune response in the recipient of the inducible pro-apoptotic polypeptides of this disclosure, cells containing the inducible pro-apoptotic polypeptides, compositions containing the inducible pro-apoptotic polypeptides, or cells containing the inducible pro-apoptotic polypeptides.

[0259] This disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, wherein the pro-apoptotic polypeptide does not contain a non-human sequence. In certain embodiments, the non-human sequence includes a restricting site. In certain embodiments, the pro-apoptotic peptide is a caspase polypeptide. In certain embodiments, the caspase polypeptide is a caspase-9 polypeptide. In certain embodiments, the caspase-9 polypeptide is a truncated caspase-9 polypeptide. The pro-apoptotic polypeptides of this disclosure may be non-spontaneously occurring.

[0260] The caspase polypeptides of this disclosure include, but are not limited to, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. The caspase polypeptides of this disclosure include, but are not limited to, apoptosis-related caspase polypeptides, including caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. The caspase polypeptides of this disclosure include, but are not limited to, apoptosis-initiating caspase polypeptides, including caspase 2, caspase 8, caspase 9, and caspase 10. The caspase polypeptides of this disclosure include, but are not limited to, apoptosis-executing caspase polypeptides, including caspase 3, caspase 6, and caspase 7.

[0261] The caspase polypeptides of this disclosure may be encoded by amino acids or nucleic acid sequences having one or more modifications compared to wild-type amino acids or nucleic acid sequences. The nucleic acid sequences encoding the caspase polypeptides of this disclosure may be codon-optimized. One or more modifications to the amino acids and / or nucleic acid sequences of the caspase polypeptides of this disclosure may increase the interactions, crosslinking, cross-activation, or activation of the caspase polypeptides of this disclosure compared to wild-type amino acids or nucleic acid sequences. Alternatively, or in addition, one or more modifications to the amino acids and / or nucleic acid sequences of the caspase polypeptides of this disclosure may decrease the immunogenicity of the caspase polypeptides of this disclosure compared to wild-type amino acids or nucleic acid sequences.

[0262] The caspase polypeptides of this disclosure may be shortened compared to wild-type caspase polypeptides. For example, the caspase polypeptide may be shortened to eliminate the sequence encoding the caspase activation and recruitment domain (CARD) in order to eliminate or minimize the possibility of activating a local inflammatory response in addition to initiating apoptosis in cells containing the inducible caspase polypeptide of this disclosure. The nucleic acid sequence encoding the caspase polypeptide of this disclosure may be spliced ​​to form a variant amino acid sequence of the caspase polypeptide of this disclosure compared to the wild-type caspase polypeptide. The caspase polypeptide of this disclosure may be encoded by recombinant and / or chimeric sequences. The recombinant and / or chimeric caspase polypeptide of this disclosure may comprise sequences from one or more different caspase polypeptides. Alternatively, or in addition, the recombinant and / or chimeric caspase polypeptide of this disclosure may comprise sequences from one or more species (e.g., human and non-human sequences). The caspase polypeptide of this disclosure may be non-spontaneous.

[0263] The ligand-binding region of the inducible pro-apoptotic polypeptide of this disclosure may contain any polypeptide sequence that promotes or facilitates the dimerization of the first inducible pro-apoptotic polypeptide of this disclosure with the second inducible pro-apoptotic polypeptide of this disclosure, the dimer which activates or induces crosslinking of the pro-apoptotic polypeptide and the initiation of apoptosis in cells.

[0264] The ligand-binding ("dimerization") region may include any polypeptide or its functional domain that enables induction using a natural or non-natural ligand (i.e., an inducer), such as a non-natural synthetic ligand. Depending on the nature of the inducible pro-apoptotic polypeptide and the choice of ligand (i.e., inducer), the ligand-binding region may be inside or outside the cell membrane. A wide variety of ligand-binding polypeptides and their functional domains, including receptors, are known. The ligand-binding regions of this disclosure may include one or more sequences from receptors. Of particular interest are ligand-binding regions for which the ligand (e.g., a small organic ligand) is known or readily available. These ligand-binding regions or receptors may include, but are not limited to, FKBP and cyclophylline receptors, steroid receptors, tetracycline receptors, and "non-natural" receptors (which can be obtained from antibodies, particularly heavy or light chain subunits, their variant sequences, probabilistic procedures, combinatorial synthesis, and other random amino acid sequences). In a particular embodiment, the ligand-binding region is selected from the group consisting of an FKBP ligand-binding region, a cyclophylline receptor ligand-binding region, a steroid receptor ligand-binding region, a cyclophylline receptor ligand-binding region, and a tetracycline receptor ligand-binding region.

[0265] The ligand-binding region, containing one or more receptor domains, may consist of at least about 50 amino acids and less than about 350 amino acids, typically less than 200 amino acids, either as the native domain or its truncated active portion. The binding region may be a small (less than 25 kDa, to enable efficient transfection in viral vectors), monomeric, non-immunogenic, readily available synthetically, cell-permeable, and non-toxic ligand, which may be configured for dimerization, for example.

[0266] The ligand-binding region, containing one or more receptor domains, may be intracellular or extracellular, depending on the design of the inducible pro-apoptotic polypeptide and the availability of a suitable ligand (i.e., inducer). For hydrophobic ligands, the binding region may be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region is usually outside the cell membrane unless a transport system exists to transport the ligand internally in a form available for binding. For intracellular receptors, the inducible pro-apoptotic polypeptide, or a transposon or vector containing the inducible pro-apoptotic polypeptide, may encode a signal peptide and a transmembrane domain on the 5' or 3' side of the receptor domain sequence, or may have a lipid adhesion signal sequence on the 5' side of the receptor domain sequence. When the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain is extracellular.

[0267] Antibodies and antibody subunits, such as heavy or light chains, specifically fragments, more specifically, all or part of their variable region, or heavy-light chain fusions that produce high-affinity binding, can be used as ligand-binding regions in this disclosure. Antibodies intended include ectopically expressed human products, such as extracellular domains, that do not evoke an immune response and are generally not expressed in the periphery (i.e., outside the CNS / brain region). Such examples include, but are not limited to, low-affinity nerve growth factor receptors (LNGFRs) and embryonic surface proteins (i.e., carcinoembryonic antigens). Furthermore, antibodies can be prepared for physiologically acceptable hapten molecules, and their individual antibody subunits can be screened for binding affinity. The cDNA encoding the subunit can be isolated and modified, such as by deletion of a portion of the constant region or variable region, or by mutagenesis of the variable region, to acquire a binding protein domain with appropriate affinity for the ligand. Thus, almost any physiologically acceptable hapten compound can be used as a ligand or to provide an epitope for a ligand. Instead of an antibody unit, an endogenous receptor can be used, which has a known binding region or domain and for which a useful or known ligand for binding exists.

[0268] With respect to the polymerization of receptors, a ligand for the ligand-binding region / receptor domain of an inducible pro-apoptotic polypeptide can be a polymer in the sense that the ligand may have at least two binding sites, each of which has the ability to bind to a ligand-receptor domain (i.e., a ligand having a first binding site capable of binding to the ligand-binding region of a first inducible pro-apoptotic polypeptide, and a second binding site capable of binding to the ligand-binding region of a second inducible pro-apoptotic polypeptide, wherein the ligand-binding region of the first inducible pro-apoptotic polypeptide and the ligand-binding region of the second inducible pro-apoptotic polypeptide are either identical or different). Therefore, as used herein, the term “multimeric ligand-binding region” refers to the ligand-binding region of an inducible pro-apoptotic polypeptide of this disclosure that binds to a polymeric ligand. Multimeric ligands of this disclosure include dimeric ligands. Dimeric ligands of this disclosure may have two binding sites capable of binding to a ligand-receptor domain. In certain embodiments, the multimer ligands of this disclosure are dimers or higher-order oligomers of synthetic organic small molecules, typically about tetramers or less, and the individual molecules are typically at least about 150 Da, less than about 5 kDa, and usually less than about 3 kDa. Various pairs of synthetic ligands and receptors can be used. For example, in embodiments including innate receptors, dimerized FK506 can be used with the FKBP12 receptor, dimerized cyclosporine A can be used with the cyclophylline receptor, dimerized estrogen can be used with the estrogen receptor, dimerized glucocorticoid can be used with the glucocorticoid receptor, dimerized tetracycline can be used with the tetracycline receptor, dimerized vitamin D can be used with the vitamin D receptor, and so on. Alternatively, higher-order ligands, such as trimers, can be used.In embodiments including non-natural receptors, such as antibody subunits, modified antibody subunits, single-chain antibodies consisting of heavy and light chain variable regions separated by a flexible linker, or modified receptors, and their variant sequences, a wide variety of compounds can be used. An important characteristic of the units containing the multimeric ligands of this disclosure is that each binding site can bind to the receptor with high affinity, and preferably, they can be chemically dimerized. Furthermore, methods are available to maintain the hydrophobic / hydrophilic balance of the ligands so that they can dissolve in serum at a functional level for most applications and further diffuse across the plasma membrane.

[0269] Activation of the inducible pro-apoptotic polypeptides of this disclosure can be achieved, for example, through inducer-mediated chemoinducible dimerization (CID) to produce conditionally regulated proteins or polypeptides. The pro-apoptotic polypeptides of this disclosure are not only inducible, but their induction is also reversible, resulting from the degradation of unstable dimerizing agents or the administration of monomer competitive inhibitors.

[0270] In certain embodiments, the ligand-binding region comprises the FK506-binding protein 12 (FKBP12) polypeptide. In certain embodiments, the ligand-binding region comprises the FKBP12 polypeptide having a phenylalanine (F) to valine (V) substitution at position 36 (F36V). In certain embodiments, the inducer may include the synthetic drug AP1903 (CAS index name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propyridene]] ester, [2S-[1(R*),2R*[S*[S*[1(R*),2R*]]]]]-(9Cl) CAS registry number: 195514-63-7; molecular formula: C78H98N4O20; molecular weight: 1411.65)). In certain embodiments, the inducer may include AP20187 (CAS registry number: 195514-80-8 and molecular formula: C82H107N5O20), which comprises the FKBP12 polypeptide having a ligand-binding domain with a phenylalanine (F) to valine (V) substitution at position 36 (F36V). In certain embodiments, the inducer may be an AP20187 analog, such as AP1510. As used herein, the inducers AP20187, AP1903, and AP1510 may be used interchangeably.

[0271] AP1903 API is manufactured by Alphora Research Inc., and AP1903 injectable formulation is manufactured by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 in a 25% solution of the nonionic solubilizer Solutol HS15 (250 mg / mL, BASF). At room temperature, the formulation is a clear, pale yellow solution. Upon refrigeration, the formulation undergoes a reversible phase transition, producing an emulsion. This phase transition is reversed by warming it back to room temperature. The filling is 2.33 mL in a 3 mL glass vial (approximately 10 mg of AP1903 for injection in total per vial). Once the need for AP1903 administration is determined, the patient may be administered a single fixed dose of AP1903 for injection (0.4 mg / kg) over 2 hours by IV infusion, for example, using an infusion set sterile with non-DEHP, non-ethylene oxide. The AP1903 dose is calculated individually for each patient and is not recalculated unless body weight fluctuates by ≥10%. The calculated dose is diluted in 100 mL of 0.9% saline before infusion. In a previous Phase I trial of AP1903, 24 healthy volunteers were treated with a single dose of AP1903 for injection at dose levels of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, and 1.0 mg / kg, administered intravenously over 2 hours. Plasma levels of AP1903 were directly proportional to the dose, with mean Cmax values ​​of approximately 10–1275 ng / mL for the dose range of 0.01–1.0 mg / kg. After the initial infusion, blood concentrations showed a rapid distribution phase, with plasma levels decreasing to approximately 18%, 7%, and 1% of the maximum concentration at 0.5 mg / kg, 2 hours, and 10 hours post-administration, respectively. AP1903 for injection was shown to be safe and well-tolerated at all dose levels, and a favorable pharmacokinetic profile was demonstrated. Iuliucci JD, et al., J Clin Pharmacol. 41:870-9, 2001.

[0272] For example, a fixed dose of AP1903 for injection used may be 0.4 mg / kg administered intravenously over 2 hours. The amount of AP1903 required in vitro for effective cell signaling is 10–100 nM (1600 Da MW). This is equivalent to 16–160 μg / L or

number

[0273] The amino acid and / or nucleic acid sequences encoding ligand binding in this disclosure may contain one or more modified sequences compared to the wild-type amino acid or nucleic acid sequence. For example, the amino acid and / or nucleic acid sequence encoding the ligand-binding region in this disclosure may be a codon-optimized sequence. One or more modifications may increase the binding affinity of a ligand (e.g., an inducer) to the ligand-binding region of this disclosure compared to the wild-type polypeptide. Alternatively, or in addition, one or more modifications may decrease the immunogenicity of the ligand-binding region of this disclosure compared to the wild-type polypeptide. The ligand-binding region and / or inducers of this disclosure may be non-spontaneously occurring.

[0274] The inducible pro-apoptotic polypeptides of this disclosure comprise a ligand-binding region, a linker, and a pro-apoptotic peptide, and the inducible pro-apoptotic polypeptides do not contain a non-human sequence. In certain embodiments, the non-human sequence includes a restriction site. The linker is a dimerization of the ligand-binding region and may include any organic or inorganic material that enables interaction, crosslinking, cross-activation, or activation of the pro-apoptotic polypeptide so that the interaction or activation of the pro-apoptotic polypeptide initiates apoptosis in the cell. In certain embodiments, the linker is a polypeptide. In certain embodiments, the linker is a polypeptide comprising a G / S rich amino acid sequence ("GS" linker). In certain embodiments, the linker is a polypeptide comprising the amino acid sequence GGGGS (SEQ ID NO: 25). In preferred embodiments, the linker is a polypeptide, and the nucleic acid encoding the polypeptide does not contain a restriction site of a restriction endonuclease. The linkers of this disclosure may be non-spontaneously occurring.

[0275] The inducible pro-apoptotic polypeptides of this disclosure may be expressed in cells under the transcriptional control of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of this disclosure in those cells. As used herein, the term “promoter” refers to a promoter that acts as the first binding site for RNA polymerase transcribing a gene. For example, the inducible pro-apoptotic polypeptides of this disclosure may be expressed in mammalian cells under the transcriptional control of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of this disclosure in mammalian cells, and such promoters include, but are not limited to, native promoters, endogenous promoters, exogenous promoters, and heterologous promoters. Preferred mammalian cells include human cells. Therefore, the inducible pro-apoptotic polypeptides of this disclosure may be expressed in human cells under the transcriptional control of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of this disclosure in human cells, and such promoters include, but are not limited to, human promoters or viral promoters. Exemplary promoters for expression in human cells include, but are not limited to, the human cytomegalovirus (CMV) pre-early gene promoter, the SV40 early promoter, the Rous sarcoma virus long-terminal repeat, the β-actin promoter, the rat insulin promoter, and the glyceraldehyde-3-phosphate dehydrogenase promoter, each of which may be used to obtain high levels of expression of the inducible pro-apoptotic polypeptides of this disclosure. The use of promoters of other viruses, mammalian cells, or bacterial phages known in the art to achieve expression of the inducible pro-apoptotic polypeptides of this disclosure is similarly intended, provided that the expression level is sufficient to initiate apoptosis in the cells. By using promoters with known properties, the level and pattern of expression of the target protein after transfection or transformation can be optimized.

[0276] The selection of promoters controlled in response to specific physiological or synthetic signals can enable the inducible expression of the inducible pro-apoptotic polypeptides of this disclosure. The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system. This system is designed to enable the controlled expression of a gene of interest in mammalian cells. It consists of a tightly controlled expression mechanism that allows for more than 200-fold inducibility, even though there is virtually no basal level expression of the transgene. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter, turning on the expression of the downstream transgene and obtaining high levels of mRNA transcript. In this system, the ecdysone-responsive promoter that drives the expression of the gene of interest resides on a separate plasmid, but both monomers of the heterodimeric receptor are constitutively expressed from a single vector. Therefore, manipulating this type of system into a vector of interest may be useful. Another potentially useful inducible system is the Tet-Off® or Tet-On® system (Clontech, Palo Alto, Calif.), initially developed by Gossen and Bujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992; Gossen et al., Science, 268:1766-1769, 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-On® system, gene expression is turned on in the presence of doxycycline, while in the Tet-Off® system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements derived from the E. coli tetracycline resistance operon: a tetracycline operator sequence (to which the tetracycline repressor binds) and a tetracycline repressor protein.The target gene is cloned into the plasmid behind a promoter where a tetracycline-responsive element is present. A second plasmid contains a regulatory element called a tetracycline-regulatory transactivator, which in the Tet-Off® system consists of a VP16 domain from herpes simplex virus and a wild-type tetracycline repressor. Thus, in the absence of doxycycline, transcription is constitutively turned on. In the Tet-On® system, the tetracycline repressor activates transcription in the presence of doxycycline, rather than the wild-type. The Tet-Off® system can be used when creating gene therapy vectors, so that producing cells can grow in the presence of tetracycline or doxycycline and potentially inhibit the expression of toxic transgenes, although gene expression may be constitutively turned on when the vector is introduced into a patient.

[0277] In some situations, it is desirable to regulate the expression of the transgene in a gene therapy vector. For example, different viral promoters with varying levels of activity are used depending on the desired expression level. In mammalian cells, the CMV pre-initial promoter is often used to produce potent transcriptional activation. The CMV promoter is outlined in Donnelly, JJ, et al., 1997. Annu. Rev. Immunol. 15:617-48. When a low level of transgene expression is desired, less potent modified versions of the CMV promoter are also used. When transgene expression is desired in hematopoietic cells, retroviral promoters such as LTRs from MLV or MMTV are often used. Other viral promoters used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as E1A, E2A, or from the MLP region, AAV LTR, HSV-TK, and aeroviral sarcoma virus.

[0278] In other cases, promoters that are developmentally regulated and active in specific differentiated cells may be selected. For example, a promoter may be inactive in pluripotent stem cells, but may be activated when, for example, pluripotent stem cells differentiate into more mature cells.

[0279] Similarly, tissue-specific promoters are used to induce transcription in specific tissues or cells to reduce potential toxicity or undesirable effects on non-target tissues. While these promoters may result in reduced expression compared to stronger promoters such as the CMV promoter, they may also result in more restricted expression and immunogenicity (Bojak, A., et al., 2002. Vaccine. 20:1975-79, Cazeaux, N., et al., 2002. Vaccine 20:3322-31). For example, tissue-specific promoters such as PSA-related promoters or prostate-specific glandular kallikrein or muscle creatine kinase genes may be used as needed.

[0280] Examples of tissue-specific or differentiation-specific promoters include, but are not limited to, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin (muscle), elastase-1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin (differentiating cells, healing tissues), and Flt-1 (endothelial cells) and GFAP (astrocytic cells).

[0281] In certain indications, it is desirable to activate transcription at a specific time point after administration of the gene therapy vector. This is done using promoters such as those modulated by hormones or cytokines. Cytokine and inflammatory protein-responsive promoters that can be used include K and T kininogen (Kageyama et al., (1987) J. Biol. Chem., 262, 2345-2351), c-fos, TNF-alpha, C-reactive protein (Arcone, et al., (1988) Nucl. Acids Res., 16(8), 3195-3207), haptoglobin (Oliviero et al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C / EBP alpha, IL-1, IL-6 (Poli and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86, 8202-8206), complement C3 (Wilson et al. al., (1990) Mol. Cell. Biol., 6181-6191), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol Cell Biol, 8, 42-51), alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell. Biol., 2394-2401, 1988), angiotensinogen (Ron, et al.) Al., (1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol ester, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (inducible by phorbol ester and retinoic acid), metallothionein (inducible by heavy metals and glucocorticoids), stromelysin (inducible by phorbol ester, interleukin-1, and EGF), alpha-2 macroglobulin, and alpha-1 anti-chymotrypsin. Other promoters include, for example, SV40, MMTV, human immunodeficiency virus (MV), Molony virus, ALV, Epstein-Barr virus, Roussarcoma virus, human actin, myosin, hemoglobin, and creatine.

[0282] Depending on the desired effect, any of the above promoters may be useful alone or in combination with other promoters. Promoter and other regulatory elements are selected so that they function effectively in the desired cells or tissues. Furthermore, this list of promoters should not be construed as exhaustive or restrictive, and other promoters may be used in conjunction with the promoters and methods disclosed herein. [Examples]

[0283] Characterization of Example 1-P-BCMA-101 (A / K / A anti-BCMA CARTyrin (A08)) CARTyrin expression in this disclosure was evaluated after mRNA electroporation of the CARTyrin-encoding sequence into T cells. The functionality of CARTyrin-expressing T cells was measured by degranulation of tumor cells. Characterization further assayed the correlation with functionality.

[0284] Figure 4 shows the structure of A08 anti-BCMA CARTyrin.

[0285] Figures 5-8 show the in vitro and in vivo characterization of P-BCMA-101 (encoding A08 anti-BCMA CARTyrin).

[0286] In vitro evaluation of A08 CARTyrin demonstrated high levels of surface expression in human primary T cells after lentiviral transduction and potent cytotoxic activity (e.g., proliferation) against BCMA+ tumor cells (see Figures 5A-C). Following this potent in vitro performance, we evaluated A08 CARTyrin's ability to function in vivo.

[0287] Figure 6 shows the treatment schedule for an in vivo study in mice using A08 CARTyrin. The results of this study show that 100% of the mice treated with P-BCMA-101 (encoding A08 CARTyrin) survived until day 21 (see Figure 7). This complete survival of the treated animals at day 21 was accompanied by zero tumor burden (assessed by M protein levels, which were not detected in these animals at day 21) (see Figure 7). Figure 8 provides a series of photographs further showing tumor burden in control animals and animals treated with P-BCMA-101. Animals expressing A08 CARTyrin show a reduction in tumor burden compared to controls.

[0288] Example 2 - Expression and function of PiggyBac with iC9 safety switch incorporated into human pan-T cells Human pan-T cells were nucleofected with one of four piggyBac transposons using an Amaxa 4D nucleofector. Modified T cells, which were to undergo a "mock" state, were nucleofected with an empty piggyBac transposon. Modified T cells received either a piggyBac transposon containing only the therapeutic agent (a sequence encoding CARTyrin) or a piggyBac transposon containing both an integrated iC9 sequence and the therapeutic agent (a sequence encoding CARTyrin).

[0289] Figure 8 provides a schematic diagram of the iC9 safety switch, including the ligand-binding region, linker, and cleaved caspase 9 polypeptide. Specifically, the iC9 polypeptide includes a ligand-binding region containing the FK506-binding protein 12 (FKBP12) polypeptide, which includes a substitution of phenylalanine (F) to valine (V) at position 36 (F36V). The FKBP12 of the iC9 polypeptide is encoded by an amino acid sequence including GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 23). The iC9 polypeptide FKBP12 is encoded by a nucleic acid sequence containing GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCTTCGATGTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 24). The linker region of the iC9 polypeptide is encoded by a nucleic acid sequence containing amino acids GGGGS (SEQ ID NO: 25) and GGAGGAGGAGGATCC (SEQ ID NO: 26).The nucleic acid sequence encoding the linker region of the iC9 polypeptide is encoded by an amino acid sequence including GFGDVGALESLRGNADLAYISLMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCNFLRKKLFFKTS (SEQ ID NO: 27).iC9 polypeptide is coded by nucleic acid sequence containing ド(sequence number 28).

[0290] To test the iC9 safety switch, each of the four modified T cells was incubated for 24 hours with 0, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM of AP1903 (an inducer of AP1903). Viability was assessed by flow cytometry using the fluorescent intercalator 7-aminoactinomycin D (7-AAD) as a marker for cells undergoing apoptosis.

[0291] Cell viability was assessed on day 12 (see Figure 9). The data show a shift in the cell population from the lower right quadrant to the upper left quadrant with increasing inducer concentration in cells containing the iC9 construct, but this effect was not observed in cells lacking the iC9 construct (those receiving only CARTyrin), and the cells were evenly distributed in these two regions regardless of inducer concentration. Furthermore, cell viability was assessed on day 19 (see Figure 10). The data reveals the same trend as shown in Figure 9 (day 12 after nucleofection), however, the population shift to the upper left quadrant is more pronounced at this later point (day 19 after nucleofection).

[0292] The aggregated results were quantified and are shown in Figure 11. The significant effect of the iC9 safety switch on cell viability is shown as a function of the concentration of the iC9 switch inducer (AP1903) in each modified cell type, on day 12 (Figure 9 and left graph) or day 19 (Figure 10 and right graph). The presence of the iC9 safety switch induces apoptosis in the majority of cells by day 12, and this effect is even more dramatic by day 19.

[0293] The results of this study demonstrate that the iC9 safety switch is highly effective in eliminating activated cells upon contact with an inducer (e.g., AP1903), as AP1903 induces apoptosis even at the lowest concentration tested (0.1 nM). Furthermore, the iC9 safety switch can be functionally expressed as part of a tricistronic vector.

[0294] Example 3: An open-label, multicenter, phase 1 trial evaluating the safety of P-BCMA-101 in patients with relapsed / refractory multiple myeloma (MM), followed by a phase 2 response and safety (prime) evaluation. Investigational drug names: P-BCMA-101 and Limiducide Autologous CAR-T cells engineered to contain anti-B cell maturation antigen (BCMA) centirin (CARTyrin) bound to the TCRζ and 4-1BB signaling domains.

[0295] Brief description of the investigational drug: P-BCMA-101 consists of genetically modified T cells using an electroporation-based nonviral (DNA transposon) gene delivery system called piggyBac® (PB) DNA modification system, which efficiently transfers DNA from plasmids to chromosomes via a "cut and paste" mechanism. Compared to lentiviral or Y-retroviral transduction, PB offers advantages such as a safer insertion profile, higher levels of transgene expression, more stable and longer-lasting transgene expression, and a highly enriched and favorable T stem cell memory (Tscm) phenotype.

[0296] P-BCMA-101 cells are autologous T cells isolated from a subject and modified to contain three main components: the anti-BCMA centintine chimeric antigen receptor (CARTyrin) gene, the dihydrofolate reductase (DHFR) resistance gene, and the inducible caspase 9 (iC9)-based safety switch gene.

[0297] The CARTyrin expression cassette encodes an extracellular BCMA-binding centiririn protein fused to the CD8a signaling / leader peptide, CD8a hinge / spacer, CD8a transmembrane domain, intracellular 4-1 BB signaling domain, and T cell receptor (TCR) ζ chain signaling domain (see, e.g., Figure 4). Compared to binding domains consisting of antibody-based single-chain variable regions (scFv), the CARTyrin-binding domain is a complete human protein, smaller, more stable, and potentially less immunogenic, yet possesses similar antigen-binding properties that enable specific recognition and killing of BCMA-expressing MM cells. CARTyrins are also designed to avoid T cell depletion.

[0298] The DHFR selection gene is used during manufacturing for ex vivo selection of transposed P-BCMA-101 T cells, resulting in a more homogeneous product.

[0299] The “safety switch” is an additional safety feature not found in most CAR-T cells, designed to allow for the rapid removal of P-BCMA-101 cells by intravenous administration of limituside, an activator and synthetic small molecule dimerizer, when directed (see Section 15.3).

[0300] Test product, dosage, and route of administration: Phase 1: Single dose – Dose levels of P-BCMA-101 administered intravenously as a single dose. Dose levels will be tested by a cohort in a 3+3 dose escalation design as described in the study design.

[0301] Phase 1: Cycle administration – Multiple doses of P-BCMA-101 administered intravenously over two 2-week cycles (Cohorts A and C) or three 2-week cycles (Cohort B). The total dose administered follows a 3+3 design starting at ≤ Maximum Tolerable Dose (MTD) determined during single-dose escalation. In both Cohorts A and B, 1 / 3 of the total dose is administered in the first cycle. In Cohort A, up to 2 / 3 of the total dose is administered in the second cycle. In Cohort B, up to 1 / 3 of the total dose is administered in the second and third cycles, respectively. In Cohort C, up to 2 / 3 of the total dose is administered in the first cycle, and up to 1 / 3 of the total dose is administered in the second cycle.

[0302] Phase 1: Combination therapy - P-BCMA-101 is administered in combination with approved therapies: lenalidomide (Cohort R: 10-25 mg orally daily for 21 days every 28 days, starting one week before P-BCMA-101 infusion, and Cohort RP: 10-25 mg orally daily for 21 days every 28 days, starting one week before apheresis and then one week before P-BCMA-101 infusion) and rituximab (Cohort RIT: 375 mg / m² 12 and 5 days before P-BCMA-101 infusion, and then every 8 weeks thereafter) and rituximab (Cohort RIT: 375 mg / m² 12 and 5 days before P-BCMA-101 infusion, and then every 8 weeks thereafter). 2 The dose of P-BCMA-101 administered follows a 3+3 design, starting from ≤MTD, which is determined during dose escalation.

[0303] Phase 2: cells 6~15×10 6 P-BCMA-101 is administered intravenously as a total dose of 1 / kg.

[0304] Limiducide may be administered intravenously at a dose of 0.4 mg / kg when clinically indicated.

[0305] Subjects who meet the criteria in Section 15.4 may be eligible to receive another infusion of P-BCMA-101.

[0306] Control therapy: None

[0307] Main Objectives: The main objectives of this examination are as follows:

[0308] The safety and maximum tolerated dose (MTD) of Phase 1-P-BCMA-101 will be determined based on dose-limiting toxicity (DLT).

[0309] To evaluate the safety and efficacy of Phase 2-P-BCMA-101.

[0310] Primary endpoints:

[0311] Phase 1 - Number of subjects with DLT at each dose level defining the MTD

[0312] Phase 2 - Safety and Tolerability Based on Adverse Events (AEs), Laboratory Tests, and Standard Clinical Tests

[0313] Overall response rate (ORR) and duration of response (DOR) as assessed by an independent review committee (IRC) according to the International Myeloma Working Group Criteria (Kumar, 2016).

[0314] Secondary Objectives: The secondary objectives of this test are to evaluate the following:

[0315] Phase 1 - Safety and feasibility of P-BCMA-101, antimyeloma efficacy of P-BCMA-101, and cell dose efficacy leading to dose selection for further evaluation in Phase 2 / 3 trials.

[0316] Phase 2 - Incidence and severity of cytokine release syndrome (CRS), and additional efficacy endpoints.

[0317] Secondary endpoints: The following secondary endpoints will be evaluated.

[0318] Phase 1 - Ability to produce P-BCMA-101 at doses prohibited by the protocol; safety and tolerability based on adverse events, laboratory tests, and standard clinical laboratory tests; CRS graded using Lee criteria (Lee, 2014); efficacy based on the International Myeloma Working Group (IMWG) Uniform Response Criteria (Rajkumar, 2011, Kumar, 2016, Cavo, 2017); overall response rate (ORR); time to response (TTR); duration of response (DOR); progression-free survival (PFS); and overall survival (OS).

[0319] CRS graded using Phase 2 Lee criteria (Lee, 2014); percentage of IL-6 antagonist, corticosteroid, and limituside use; negativity rates for OS, PFS, TTR, and minimal residual disease (MRD).

[0320] Exploratory Objectives: The exploratory objectives of this study are to do the following:

[0321] Phase 1 - Evaluating the relationship between MM plasma cell BCMA expression, circulating soluble BCMA, and clinical response.

[0322] Phase 1 and Phase 2: Characterize the expansion and functional persistence of P-BCMA-101 cells; evaluate the relationship between putative CRS markers and efficacy or safety; evaluate the effect of limituside on P-BCMA-101-related adverse events, where indicated.

[0323] Exploratory endpoints: The following exploratory endpoints will be evaluated during the trial.

[0324] Phase 1 - BCMA and / or other biomarkers in bone marrow, soluble BCMA and / or other biomarker levels in blood

[0325] Phase 1 and Phase 2 - P-BCMA-101 cells (e.g., vector copies / mL in blood and bone marrow of P-BCMA-101 cells); P-BCMA-101 cell subset composition and clonality; CRS markers: C-reactive protein (CRP), ferritin, IL-6, IL-2, TNF-α, interferon-gamma (IFN-γ)

[0326] Subject population and number: Adults with confirmed relapsing / refractory malformation measles. Up to approximately 120 subjects are planned for Phase 1. Approximately 100 evaluable subjects will be treated in Phase 2.

[0327] research design The trial will be conducted in multiple phases: Phase 1, open-label, single-dose escalation (SAD) phase; Phase 1, multiple-dose, cyclical phase; Phase 1, combination therapy with lenalidomide or rituximab phase; and Phase 2, open-label, efficacy, and safety phase, in adult subjects with relapsing / refractory malformations.

[0328] Only institutions with the resources to manage the types of acute emergencies expected with chimeric antigen receptor (CAR)-T cell administration and experience in managing oncology subjects and stem cell / bone marrow transplants will be selected to participate in this trial. The safety committee will meet regularly and review data throughout the trial.

[0329] Subjects who meet the protocol's enrollment criteria will be eligible for enrollment in the trial. After enrollment, leukocyte apheresis will be performed to obtain peripheral blood mononuclear cells (PBMCs) which will be sent to a manufacturing facility to produce P-BCMA-101 CARTyrin-T cells. The cells will then be returned to the investigational site and administered to subjects after a standard chemotherapy-based conditioning regimen, as described below.

[0330] Phase 1 – Phase 1 of the study consists of an open-label, multicenter, single-dose escalation (SAD), multi-cohort study; a multi-dose cycle cohort study; and a combination dosing study, involving up to approximately 120 adult subjects. Phase 1 of the study is planned to follow a 3+3 design for dose escalation cohorts, with three subjects in each cohort receiving P-BCMA-101 T cells (Table 1). The Safety Committee may recommend enrolling additional subjects in a cohort to further evaluate the results observed at that dose level. For each of the first two cohorts, the first three subjects will be alternately administered. If a Grade 3 related toxicity, CRS, or DLT is reported, the Safety Committee will review the data and decide whether to proceed to the next subject. The Safety Committee will review the data at the end of each cohort to decide whether to proceed to the next cohort. At the discretion of the Safety Committee, a third cohort may be initiated, with the first and second subjects in each cohort alternating.

[0331] DLT is defined as any event of National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI CTCAE) ≥ Grade 3 that involves uncontrolled proliferation of P-BCMA-101 cells and is not attributable to an underlying disease or lymphodepletion chemotherapy regimen, but is at least potentially related to P-BCMA-101, with the exception of the following: Grade 3 or 4 neutropenia with or without neutropenic fever, resolving within 28 days of the last P-BCMA-101 cell infusion. Grade 3 overheating Grade 3 or 4 thrombocytopenia resolved within 28 days of the last P-BCMA-101 cell infusion, regardless of the presence or absence of bleeding due to thrombocytopenia. Grade 3 or 4 anemia and lymphopenia Grade 3 or 4 hypogammaglobulinemia Alopecia Grade 3 or 4 nausea, vomiting, or diarrhea that responds to medical treatment within 24 hours. Immediate-type hypersensitivity reactions (fever, rash, bronchospasm) occurring within 2 hours of cell infusion (related to cell infusion), reversible to grade 2 or less within 6 hours of cell administration accompanied by standard antihistamine-based therapy. Grade 3 encephalopathy that recovers to less than Grade 2 within 28 days Grade 3 CRS according to Lee Criteria (Lee, 2014) to be resolved within 14 days. Grade 3 non-hematological abnormalities that resolve to ≤ Grade 2 within 14 days Grade 4 non-hematological abnormalities that resolve to ≤ Grade 2 within 7 days [Table 4]

[0332] 1. MTD - Maximum dose at which 1 or fewer out of 6 treated subjects showed DLT The 3+3 dose escalation is implemented as follows, with the Safety Committee reviewing data at the end of each cohort to determine the outcome: Starting in Cohort 1, at least 3 subjects are administered in the cohort. If no dose-limiting trials (DLTs) are observed in the first 3 subjects by 28 days after the final dose, the escalation may proceed to the next cohort. If DLTs are observed in one of the first 3 subjects, at least 3 additional subjects are treated at this dose level. If no further DLTs are observed, the escalation may proceed. If DLTs are observed in 2 or more of the 3 or 6 subjects, the MTD is considered to be the next lowest dose level, and further enrollment may be at an even lower dose level, or an intermediate dose level may be tested at the discretion of the Safety Committee. In events where 2 or more subjects in Cohort 1 experience DLTs, the Safety Committee may, after reviewing the available data, choose to administer 3 subjects in Cohort-1 using the same 3+3 expansion rule. In events where two or more subjects experience a DLT in Cohort-1, the Safety Committee may, considering safety and efficacy data, evaluate the risk-benefit ratio and choose to administer the three subjects at a lower dose using the same 3+3 expansion rule, or discontinue the study.

[0333] The proposed dosage (1 cell P-BCMA-101 / kg / dose) is: Cohort-1:0.25×10 6 Cohort 1: 0.75 × 10 6 Cohort 2: 2 x 10 6 Cohort 3: 6 x 10 6 Cohort 4:10 x 10 6 Cohort 5:15 x 10 6 including

[0334] Additional subjects may be administered to that cohort at the direction of the Safety Committee, based on safety and efficacy data from that cohort, to further evaluate the effects of P-BCMA-101 from that cohort, but the dose will not exceed the MTD. If cohort 5 is completed without concluding an overall MTD, the Safety Committee will administer 5-10 × 10 cells of P-BCMA-101. 6 Further escalation of the cohort can be evaluated with increments of individuals / kg.

[0335] Phase 1 - Cycle Dosing: In the Phase 1 cycle dose-dosing portion of the study, multiple doses of P-BCMA-101 are administered intravenously over two 2-week cycles (Cohorts A and C) or three 2-week cycles (Cohort B). The total dose administered follows a 3+3 design, starting with ≤MTD determined during single-dose dose escalation. In the first cycle in both Cohorts A and B, one-third of the total dose is administered. In Cohort A, up to two-thirds of the total dose is administered in the second cycle. In Cohort B, up to one-third of the total dose is administered in the second and third cycles, respectively. In Cohort C, up to two-thirds of the total dose is administered in the first cycle, and up to one-third of the total dose is administered in the second cycle. The same 3+3 dose escalation and / or tapering rules as described for single-dose administration are used. These procedures are detailed in Section 15.5.

[0336] Phase 1 - Combination Therapy: P-BCMA-101 is administered in combination with the approved therapies: lenalidomide (Cohort R and Cohort RP) and rituximab (Cohort RIT). The dose of P-BCMA-101 administered follows a 3+3 design, starting at 5 MTD, which will be determined during dose escalation. The same 3+3 dose escalation and / or tapering rules as described for single doses are used. These procedures are detailed in Section 15.6.

[0337] Phase 2 – Phase 2 of the trial is an open-label, multicenter study involving approximately 100 adult subjects with relapsing and / or refractory MM. Subjects were given 6–15 × 10 cells. 6 The total dose is received in units / kg (according to the schedule determined in Phase 1).

[0338] Study visits – Subjects treated in Phase 1 and Phase 2 will undergo a series of measurements of safety, tolerability, and response (myeloma staging). These measurements will be taken at screening, enrollment, or baseline visits, and during the conditioning chemotherapy period. Follow-up visits for both Phase 1 and Phase 2, as well as day 10, weeks 2, 3, 4, 6, and 8, and months 3, 4, 5, 6, 7, 8, and 9, and then every 3 months for up to 24 months after P-BCMA-101 administration. After completing or discontinuing this protocol, consenting subjects receiving P-BCMA-101 should be enrolled in a separate protocol that allows for continuous follow-up for a total of 15 years after the last dose to assess long-term safety.

[0339] Screening visit - Participants who consent will undergo a screening visit to determine their eligibility. Participants who meet all inclusion criteria and none of the exclusion criteria will return for registration and leukocytosis.

[0340] Enrollment Visit - Eligible subjects will return for enrollment and provide the samples and measurements that must be collected prior to leukocyte apheresis. Enrollment evaluation should be performed 14 days (±3 days) prior to leukocyte apheresis or with the approval of the medical monitor.

[0341] Leukocyte Apheresis Visit - Eligible subjects to be enrolled will return to the clinic for leukocyte apheresis to obtain PBMCs for P-BCMA-101 production. This visit should occur within approximately 28 days of the screening visit. Once the product is produced, subjects will return approximately 4 weeks after the leukocyte apheresis visit for combination therapy (if applicable), conditioning chemotherapy, and the P-BCMA-101 cell administration period. If P-BCMA-101 cells meeting the release criteria cannot be produced from the leukocyte apheresis sample, a second leukocyte apheresis and production may be attempted. If the second attempt also fails, the subject will be withdrawn from the study and will be considered not to have received the study treatment.

[0342] Subjects who experience rapid disease progression after leukocyte apheresis and before the conditioning chemotherapy and P-BCMA-101 cell administration period may receive salvage therapy at the discretion of the investigator. Salvage therapy should not be used unless necessary and should be determined at the discretion of the investigator, based on the subject's medical history (previously used medications are preferred and require approval from the medical monitor). If a subject receives salvage therapy, the conditioning chemotherapy and P-BCMA-101 cell administration period should be scheduled at least two weeks or five half-lives after the last treatment of salvage therapy, and the subject should meet the enrollment criteria (including those relating to measurable MM) and the criteria set out in sections 4 and 6 regarding concomitant medications. The subject's response to salvage therapy will be evaluated by the investigator and medical monitor to determine whether the subject remains eligible to receive the investigational drug.

[0343] Participants are permitted to receive radiotherapy or plasma exchange therapy, which will be exchanged for palliative purposes throughout the study period.

[0344] Baseline Visit (-12 to -6 days) - Once the product is manufactured, subjects will revisit for baseline evaluation during the week prior to initiating conditioning chemotherapy to confirm eligibility to continue. Within 72 hours 5 days prior, the following evaluations should be repeated: Minimally Invasive Mental State Examination (MMSE), physical examination, vital signs, chemistry panel including electrolytes and magnesium, hematology including B cell and T cell counts, coagulation, circulating myeloma / plasma cells, and pregnancy test (if applicable). Baseline myeloma response evaluation must be performed within 7 days of initiating conditioning chemotherapy and combination therapy. Baseline fresh samples of bone marrow and tumor are not used to confirm eligibility, and the 7-day window is intended to provide flexibility to subjects and investigators. If a new bone marrow biopsy / aspiration is performed and provided during screening, this does not need to be repeated during the baseline visit.

[0345] Conditioning chemotherapy and P-BCMA-101 cell administration period - Before administering P-BCMA-101 cell infusion, subjects received 300 mg / m² 2 Cyclophosphamide and 30 mg / m² 2 Subjects will receive a fludarabine conditioning lymphocyte depletion chemotherapy regimen, with each chemotherapy agent administered intravenously daily for three consecutive days (-5 to -3). Subjects should continue to meet the inclusion criteria or have received approval from the medical monitor at the start of conditioning chemotherapy. For subjects in Cohort R, Cohort RP, and Cohort RIT, combination therapy should be administered prior to conditioning chemotherapy on the applicable day.

[0346] Following a two-rest period after a lymphocyte-depleting chemotherapy regimen, subjects will receive P-BCMA-101 intravenously over approximately 5–20 minutes (Day 0) (subjects should be premedicated with acetaminophen and diphenhydramine). Previous studies using CAR-T therapy have shown toxicity peaks occurring 3–7 days after administration of the investigational drug. Study subjects will be closely monitored during and after infusion, and for approximately 7 days thereafter. This observation period will include an assessment of a series of AEs, including the appearance of P-BCMA-101 cytotoxicities such as CRS in all subjects. CRS will be graded using the Lee Criteria (Lee, 2014). Guidance on AE grading and management can be found in Section 8 of this protocol and in the Study Reference Manual. Guidance on the use of limituside in serious P-BCMA-101-related toxicity can be found in Section 15.3 and in the Study Reference Manual.

[0347] If the investigator determines it is appropriate based on the individual patient's risk, subjects may be admitted to hospitalization for P-BCMA-101 administration. While hospitalization is not required, subjects will remain within 50 miles of a hospital for approximately 14 days after the last dose of P-BCMA-101 and will be evaluated for hospitalization in case of symptoms of CRS or neurotoxicity such as fever. If hospitalized, subjects will not be discharged until assessed as stable by the investigator. Subjects may be maintained as hospitalized patients before P-BCMA-101 administration during lymphocyte depletion chemotherapy, or after the above criteria deemed appropriate by the investigator are met.

[0348] Follow-up visits: Day 10, 2, 3, 4, 6, 8 weeks, 3, 4, 5, 6, 7, 8, 9, 12, 15, 18, 21, and 24 months. Subjects will revisit for routine follow-up after the last dose of P-BCMA-101 to undergo a series of safety, tolerability, and anti-myeloma response assessments as specified in the event schedule.

[0349] Repeated Doses: If sufficient P-BCMA-101 cells remain from manufacture when a subject's disease progresses, additional cells may be administered, with the approval of the Safety Committee, up to the highest dose level for which dose-limiting toxicity assessments have been successfully completed. To receive additional P-BCMA-101 T cell infusions, subjects will be assigned a new subject identification number and must meet all eligibility criteria described for the initial dosing and undergo the same screening, registration, conditioning chemotherapy procedures, and follow-up procedures, excluding leukocyte apheresis. The retreatment procedure is outlined in Section 15.4.

[0350] Safety Monitoring: A safety committee, comprised of the principal investigator and the clinical representative of the sponsor, will be established to regularly review data for all subjects and each cohort to determine dose escalation and enrollment.

[0351] Inclusion criteria: 1. You must have signed a written informed consent form. 2. Male or female aged 18 or more. 3. The initial diagnosis must include a confirmed diagnosis of active MM as defined by the IMWG criteria (Rajkumar, 2014). 4. It must have a measurable MM defined by at least one of the following criteria: Phase 1: Serum M protein: 0.5 g / dL (5 g / L) or higher. Urinary M-protein: 200 mg / 24 hours or more, Serum free light chain (FLC) assay: If the serum FLC ratio is abnormal, the involved FLC level is 10 mg / dL (100 mg / L) or higher. Bone marrow plasma cells: >30% of total bone marrow cells, or other measurable bone disease (e.g., plasmacytoma measurable by PET or CT) (with approval of medical monitoring) Phase 2: Serum M protein: 1.0 g / dL (10 g / L) or higher. Urinary M-protein: 200 mg / 24 hours or more, Serum FLC assay: If the serum FLC ratio is abnormal, the involved FLC level is 10 mg / dL (100 mg / L) or higher. 5. The patient must have relapsing / refractory MM as defined below. Phase 1: At least three prior therapy lines must be received, and must include proteasome inhibitors and immunomodulators (IMiDs), or In cases of "dual refractory" to proteasome inhibitors and IMiD, patients must have received at least two prior therapy lines and the condition is defined as progression within 60 days of treatment with these drugs. Phase 2: At least three prior therapy lines, which must include a proteasome inhibitor, IMiD, and CD38-targeted therapy, have been received in a triple combination form with at least two of the prior lines, and have received ≥2 cycles of each line unless PD is the best response, and The latest treatment lines are intractable, and You are either undergoing ASCT or are not a candidate for ASCT. Note: Induction therapy, autologous stem cell transplantation (ASCT), and maintenance therapy should be considered a single line of treatment if administered consecutively without hindering disease progression. 6. Participants must intend to use contraception from the time of screening throughout the entire trial (both men and women of childbearing potential). Women in cohorts R, RP, or RIT must commit to continuously refraining from sexual intercourse or using two reliable methods of contraception, starting four weeks before the commencement of treatment, during treatment, during dose interruption, and for four weeks after discontinuation of lenalidomide and for 12 months after the final dose of rituximab. Men in Cohort R or RP, even if they have undergone a successful vasectomy, must always use latex or synthetic condoms when having sexual contact with fertile women while taking lenalidomide and for up to four weeks after discontinuing lenalidomide. Male patients taking lenalidomide must not donate sperm. 7. Screening requires a negative serum pregnancy test, and a negative urine test within 3 days prior to initiating a lymphocyte depletion chemotherapy regimen (for women of childbearing potential). Female subjects in cohorts R and RP must have two negative pregnancy tests before starting lenalidomide therapy. The first test must be performed within 10-14 days prior to the subject starting lenalidomide therapy, and the second test within 24 hours. Tests must then be performed weekly for the first month, and thereafter monthly for women with regular menstrual cycles and every two weeks for women with irregular menstrual cycles. 8. If autologous stem cell transplantation has been performed, it must be at least 90 days later. 9. It must have appropriate biological organ functions as defined below (or approved by the medical monitor): Calculated using the Cockcroft-Gault formula, with dialysis-independent serum creatinine <2.0 mg / dL and estimated creatinine clearance >30 mL / min. Absolute neutrophil count >1,000 / μL and platelet count >50,000 / μL (or >30,000 / μL if bone marrow plasma cells are >50% cellular). An appropriate absolute CD3 count (Phase 2: absolute lymphocyte count > 300 / μL) is estimated to obtain the target cell dose based on the dose cohort. Hemoglobin > 8 g / dL (Blood transfusion and / or growth factor supplementation are acceptable). Serum glutamate oxaloacetate transaminase (SGOT) < 3 × upper limit of normal, and total bilirubin < 2.0 mg / dL (in the absence of a molecularly documented history of Gilbert's syndrome). Left ventricular ejection fraction (LVEF) > 45%. LVEF assessment must be performed within 4 weeks of registration. 10. According to the NCI CTCAE version 4.03 criteria or the subject's previous baseline, the patient must have recovered to grade < 2 from toxicity due to prior treatment, excluding peripheral neuropathy. The clinical trial must have a performance status of 11.0-1 in the Eastern Cooperative Oncology Group (ECOG).

[0352] Exclusion criteria: 1. Pregnant or lactating; 2. Inadequate venous access and / or contraindications to leukocytosis; 3. Active hemolytic anemia, plasma cell leukemia, Waldenström macroglobulinemia, POEMS syndrome (polyneuropathy, organomegaly, endocrine disorders, monoclonal protein, and skin changes), disseminated intravascular coagulation, leukopenia, or amyloidosis; 4. In addition to MM, having an active second malignancy (not disease-free for at least 5 years), excluding low-risk neoplasms such as non-metastatic basal cell carcinoma or squamous cell carcinoma; 5. 6. Having an active autoimmune disease such as psoriasis, multiple sclerosis, lupus, or rheumatoid arthritis (medical monitor will determine if the disease is active and autoimmune), 7. Having a history of a serious central nervous system (CNS) disorder such as stroke or epilepsy (medical monitor will determine if it is serious), 8. Having an active systemic infection (e.g., causing fever or requiring antimicrobial treatment), 9. Having hepatitis B or C virus, human immunodeficiency virus (HIV), or human T-lymphotropic virus (HTLV) infection, or any of these immunodeficiency syndromes, 10. Having a New York Heart Association (New 10. Having a history of York Heart Association (NYHA) class III or IV heart failure, unstable angina, or myocardial infarction or a major arrhythmia (e.g., atrial fibrillation, sustained [>30-second] ventricular tachycardia, etc.); having any psychiatric or medical disorder (e.g., cardiovascular, endocrine, renal, gastrointestinal, genitourinary, immunodeficiency, or pulmonary disorder) that, in the opinion of the principal investigator or medical monitor, would hinder safe participation in and / or adherence to the protocol (including any medical condition or laboratory findings that would make it unlikely that the patient is eligible for or unable to receive appropriate leukocytosis, conditioning chemotherapy, and / or CAR-T cell administration); 11. Having previously received (or been approved by the medical monitor for) gene therapy or genetically modified cell immunotherapy.12. Subjects may have received non-modified autologous T cells or stem cells in connection with anti-myeloma treatment; 13. Subjects have received anticancer drugs within 2 weeks or 5 half-lives (whichever is longer, or with the approval of the medical monitor) of the start of conditioning chemotherapy; 14. Subjects have received immunosuppressants within 2 weeks of the start of leukocyte apheresis and / or are expected to need them during the study (the medical monitor will determine whether a drug is considered immunosuppressive). In general, all non-essential medications (including supplements, herbal medicines, etc.) should be discontinued from 2 weeks before leukocyte apheresis until 2 months after P-BCMA-101 administration, as they may not have an immunosuppressive effect; 15. Subjects have received or are expected to need systemic corticosteroid therapy of prednisone ≥ 5 mg / day or an equivalent dose of another corticosteroid within 2 weeks of the required leukocyte apheresis or within 1 week or 5 half-lives (whichever is shorter) of P-BCMA-101 administration. (Topical and inhaled corticosteroids are permitted. Systemic corticosteroids are contraindicated after receiving P-BCMA-101 cells, outside of study-specific guidance), 15. Having CNS metastases of myeloma or symptomatic CNS complications (including leptomeningeal carcinomatosis, cranial neuropathy, or mass lesions, and spinal cord compression), 16. Having a history of severe immediate-type hypersensitivity reactions to any of the drugs used in this study, 17. Having a history of allogeneic stem cell transplantation or any other allogeneic or xenotransplantation, or having undergone autologous transplantation within the last 90 days, 18. Not being able to take acetylsalicylic acid (ASA) (325 mg) daily as prophylactic anticoagulation. Patients tolerant to ASA may be given warfarin or low molecular weight heparin (Cohorts R and RP only), 19. Having a history of thromboembolic disease within the past 6 months, regardless of anticoagulation (Cohorts R and RP only).

[0353] Study duration: Participants will be followed for up to two years after the last dose of this study, after which consenting participants will transition to a long-term safety follow-up protocol for a total of 15 years of follow-up from the last dose.

[0354] Event Schedule: For single-dose events, see Table 2 for screening with conditioning chemotherapy, and for events, see Table 3 for P-BCMA-101 administration and follow-up. Event schedules for retreatment of subjects with P-BCMA-101 are listed in Tables 8 and 9 (see Section 15.4). For limitucide treatment, see the event schedule in Table 7. Event schedules for subjects in the cycle-dosing cohort are listed in Tables 10, 11, 12, and 13 (see Section 15.5). Event schedules for subjects in the combination-dosing cohort are listed in Tables 14 and 15 (see Section 15.6).

[0355] Criteria for suspending administration or discontinuing the study: If a DLT or any treatment-related death occurs as defined in the study, administration to new subjects will be suspended until the Safety Committee meets, reviews the event, and determines the subsequent plan, which may include discontinuing the study, reducing subsequent dose levels, establishing additional safety procedures or study modifications, or continuing the study or event as planned by other appropriate means. As described above, if two or more subjects have DLT in the Phase 1 cohort, and CRS with an incidence of ≥10% and ≥Grade 4 or an incidence of ≥30% and ≥Grade 3, or if >10 treated patients have neurotoxicity at or below the corresponding dose level in Phase 2, then the dose level is above the MTD, and any further administration will be at a lower dose level.

[0356] Statistical Methodology: Demographic and baseline characteristics, safety, and efficacy data are summarized using appropriate descriptive statistics. Data analysis is provided for each dose cohort, and, where necessary, for all subjects combined. Descriptive statistics, including mean, median, standard deviation, and range, are calculated for continuous variables, and categorical data are summarized using numerical and percentage values. For response rate endpoints, point estimates and two-sided binomial 95% confidence intervals are calculated. Time variables to events are summarized using the Kaplan-Meier method.

[0357] Treatment-induced adverse events (TEAEs) are summarized for all subjects combined, using the number and percentage of subjects per cohort. TEAEs are also summarized by severity and relationship. Concomitant medications are summarized for each dose cohort, using the number and percentage of subjects.

[0358] Vital signs, electrocardiogram (ECG) measurements, and laboratory results are summarized for each cohort using descriptive statistics for observed values ​​and changes from baseline values. Laboratory results are also summarized for each cohort in comparison to the normal range (less than, within, or greater than).

[0359] The Phase 1 portion of the trial is a standard 3+3 design of dose cohorts aimed at determining the dose at which the incidence of DLT is less than 33%. Therefore, up to 120 subjects may be enrolled, potentially including 18 cohorts of 6 subjects during dose escalation, cyclical administration, and concomitant administration, as well as subjects who may be enrolled to further evaluate cohort outcomes in exchange for those who discontinue before completion of DLT.

[0360] In the Phase 2 portion of the trial, the response rate endpoint is tested, excluding response rates of ≤30% obtained with p<0.05 using the recently approved standard treatment, daratumumab. The time variable to event is summarized using the Kaplan-Meier method. Using a sample of 100 subjects, the Phase 2 portion of the trial has 90% power and detects a 15 percent point improvement for a 30% response rate. This power calculation is based on a direct test of binomial proportions with a one-sided significance level of 0.05. A futility analysis is performed when 35 subjects are enrolled, receive P-BCMA-101, and are followed up for 4 months, or progress before 4 months of follow-up. This set of analyses is called the futility analysis set (FAS). In the futility analysis, a futility index (FI) equal to 1 minus the conditional power (CP) is used, based on the ratio of BORs observed in the FAS. If the FI score exceeds 0.80 (i.e., if the CP score falls below 0.20), the test may be terminated.

[0361] Subjects receiving additional P-BCMA-101 injections will also be analyzed as a separate subgroup for all subsequent outcomes. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 6-1] [Table 6-2] [Table 6-3]

[0362] Example 4 - P-BCMA-101 T-cell therapy in multiple myeloma 1. Introduction Multiple myeloma (MM) is generally an incurable and fatal disease, typically characterized by multiple relapses and recurrences. Current treatment options are inadequate, and there remains an unmet need for effective and lasting MM therapy. Chimeric antigen receptor T-cell (CAR-T) immunotherapy is emerging as a significant potential treatment approach for cancer, including MM. B-cell maturation antigen (BCMA) is an attractive target given its expression in MM cells; however, among non-malignant cells, BCMA expression is largely restricted to plasma cells and B-cell subsets. Recently, clinical data from MM patients using two similar BCMA-targeted CAR-T cell products (NCI / NIH, University of Pennsylvania, and BlueBird Bio studies) have demonstrated the safety and efficacy of this approach (Ali, 2016; Cohen, 2016; Berdeja, 2016). In vivo and in vitro studies have shown that P-BCMA-101 T cells bind to BCMA+ tumor lines with high affinity and specificity, resulting in robust degranulation and cytotoxicity. Combined with available preclinical and clinical data, as well as the potential safety and efficacy benefits of this construct, the unmet medical needs provide a rationale for evaluating P-BCMA-101 in patients with relapsed or recurrent MM. Further information is also provided in the investigator's booklet.

[0363] 1.1 Multiple Myeloma Multiple myeloma (MM) is a treatable but usually incurable plasma cell malignancy that often follows an aggressive and fatal clinical course. In 2015, an estimated 26,850 new cases of MM occurred in the United States, along with approximately 11,240 deaths. Diagnosis is most common in people in their 60s and 70s (Howlader, 2015).

[0364] A prominent feature of MM is the monoclonal proliferation of plasma cells in the bone marrow, accompanied by the overproduction of monoclonal antibodies (mAbs) that produce "M spikes" on serum protein electrophoresis (Raab, 2009). The clinical features of this disease arise from bone marrow infiltration by malignant clones, high levels of circulating monoclonal antibodies (mAbs) and / or free light chains, suppressed immunity, and peripheral organ injury. Typical signs and symptoms of MM include anemia, bleeding due to thrombocytopenia, frequent infections due to leukopenia and low antibody production, bone pain due to bone lesions and fractures, and renal impairment due to high levels of M protein accumulation and hypercalcemia.

[0365] Recent advances in understanding the pathophysiology of MM and the introduction of new therapeutic agents have contributed to the management of this disease, and a dramatic improvement in survival rates has been observed over the past 20 years. The median survival time has increased from approximately 2 years in the 1980s to 5-6 years or more today (Engelhardt, 2010). Treatment regimens tend to consist of 2-3 drugs, and almost all patients receive proteasome inhibitors (bortezomib or carfilzomib) and immunomodulatory agents (IMiDs) (lenalidomide, pomalidomide, thalidomide) both early and late in the course of treatment for these diseases. In addition, eligible patients may receive autologous hematopoietic stem cell transplantation and / or, less frequently, allogeneic hematopoietic stem cell transplantation. In 2015, several new therapies were approved in the United States, including two mAbs (daratumumab and elotuzumab), a panhistone deacetylase (HDAC) inhibitor (panabinostat), and an oral proteasome inhibitor (ixazomib). While the long-term effects of these recent approvals still need to be determined, they do not appear to produce a cure in relapsing and / or refractory settings, and most patients eventually relapse and die (Kumar, 2008). Relapsing tumors tend to recur more aggressively with each relapse.

[0366] The response to treatment is not sustained, and subsequent disease progression leads to treatment-refractory disease associated with a shortened survival time (Kumar, 2012).

[0367] 1.2 Theoretical basis for P-BCMA-101 T-cell therapy in multiple myeloma 1.2.1.P-BCMA-101 T cell therapy P-BCMA-101 is an autologous centiririn-based CAR-T cell therapy, referred to as CARTyrin T cells. These are biological products designed to target MM cells expressing the cell surface antigen BCMA, directing cytotoxic effects towards the targeted cells (Tai, 2015). The mechanism of action of P-BCMA-101 is the same as other CAR-T approaches (e.g., Ali, 2016, Berdeja, 2016). Peripheral blood mononuclear cells (PBMCs) from each patient are collected by leukocyte apheresis and then used to generate the individual patient's PBCMA-101 investigational drug via electroporation of transposer zeribonucleic acid (RNA) together with a DNA plasmid encoding a PB transposon containing CARTyrin (i.e., piggyBac® (PB) DNA modification system), followed by culture / growth.

[0368] P-BCMA-101 cells are designed to contain three key components: the anti-BCMA centintine chimeric antigen receptor (CARTyrin) gene, the dihydrofolate reductase (DHFR) resistance gene, and the inducible caspase 9 (iC9)-based safety switch gene (Hermanson, 2016).

[0369] The CARTyrin expression cassette encodes an extracellular BCMA-binding centiririn protein fused to the CD8a signaling / leader peptide, CD8a hinge / spacer, CD8a transmembrane domain, intracellular 4-1 BB signaling domain, and T cell receptor (TCR) ζ chain signaling domain. Compared to binding domains consisting of antibody-based single-chain variable regions (scFv), the CARTyrin-binding domain is a complete human protein, smaller, more stable, and potentially less immunogenic, yet possesses similar antigen-binding properties that enable recognition and killing of BCMA-expressing MM cells. This construct is also designed to avoid T cell depletion.

[0370] The DHFR resistance gene is used during T cell production for ex vivo selection of CARTyrin-expressing T cells, producing a more homogeneous product to enhance efficacy and safety.

[0371] The safety switch is an additional safety feature not found in most CAR-T cells, designed to allow for the rapid removal of P-BCMA-101 cells by intravenous administration of the synthetic dimerizing agent limituside (see Section 15.3).

[0372] As of July 14, 2019, 36 subjects were being treated with P-BCMA-101 cells (34 in Phase 1 and 2 in Phase 2), of which 3 were receiving a second dose of P-BCMA-101. All Phase 1 dose-escalation cohorts (P-BCMA-101 cells 1-5: 0.75-15 × 10) 6 The study (cells / kg / dose) was successfully completed with favorable safety and efficacy reported at the highest dose level. No DLTs were reported, and the enrollment of new subjects continues with cohort expansion, suggesting that additional cohorts should be evaluated. Patients had received numerous prior treatments (3–18 prior treatments), most of which had failed IMiD, proteasome inhibitors, daratumumab, and ASCT. Patients had a total of 48–1545 × 10¹⁶ P-BCMA-101 cells. 6 Treatment was administered at cells / kg. Circulating P-BCMA-101 cells were detected in patients' blood by PCR, and proliferation peaked at 2-3 weeks. Beyond the threshold level reached in cohorts 2-3, the response appeared to occur in patients with a broader peak, suggesting that there may be additional benefits with repeated or divided dosing. Signs of anti-CAR-T antibodies were shown in some patients, although their significance is unclear. The most common TEAEs (>30%) were neutropenia, WBC reduction, thrombocytopenia, anemia, nausea, constipation, and febrile neutropenia. Only 4 subjects had cases of CRS (one subject had 2 × 10⁶ cells). 6 The cells had a grade of 2 at cells / kg, and the three subjects had 15 × 10 cells. 6One case of CRES (6 × 10 cells / kg with grade 2), 6 Grade 2 transient confusion at cells / kg had been reported. Repeated administration of P-BCMA-101 was well tolerated. Of the Phase 1 subjects, 29 were evaluable for response according to IMWG criteria, and at least one myeloma response evaluation was completed. To date, 17 have shown a response (approximately 0.75 × 10⁶ cells). 6 1 / 2 is cells / kg, approximately 2 x 10⁶ cells 6 5 / 7 cells / kg, approximately 6 x 10⁻⁶ cells 6 Cells / kg: 4 / 9 (+2MR), approximately 10 x 10 6 Cells / kg is 3 / 4 (+1MR), and the cells are approximately 15 × 10⁻⁶. 6 (4 / 7 units / kg). Based on these results, the study was further expanded in the Phase 2 portion to further characterize safety and efficacy at dose levels 3–5.

[0373] 1.2.2. BCMA as a therapeutic target in CAR-T cells Myeloma possesses several characteristics that make it suitable for treatment with adoptive T-cell therapy. Firstly, myeloma is primarily a disease of the bone marrow, and CD19-targeted adoptive T-cell therapy has been particularly successful in myelo-dominant diseases such as acute lymphoblastic leukemia (ALL) (Brentjens, 2013). Secondly, autologous stem cell transplantation is the standard treatment for myeloma, and lymphocyte depletion can enhance the effectiveness of adoptive T-cell therapy (Brentjens, 2011, Pegram, 2012). Thirdly, unlike all other treatments for myeloma, allogeneic SCT offers the potential for a cure, but its application is limited by transplant-related toxicity, mortality, patient eligibility, and the availability of suitable donors. CAR-T cell therapy may be a safer way to achieve such antitumor efficacy (Milone, 2015).

[0374] The usefulness of CD19 as a target is limited by the fact that it is rarely expressed in malignant plasma cells of MM, and therefore other antigens have been explored, with particular attention paid to antigens that are expressed by tumor cells but not by normal tissues. One intriguing target is BCMA. The rationale for selecting this target is that although BCMA is detected in MM cells, even among non-malignant cells, BCMA expression is largely limited to a subset of plasma cells and B cells, and therefore may be targeted and less likely to have extratumor effects.

[0375] MM tumor cell recognition occurs when BCMA-specific CARs expressed on the surface of P-BCMA-101 T cells bind to BCMA antigens expressed on the surface of MM tumor cells. Signaling and activation are mediated by the cytoplasmic signaling domains 4-1BB and CD3~ encoded within the CAR. Activation can result in direct cytotoxicity of targeted MM tumors through CAR-T cell-mediated release of granzymes and perforins. Tumor killing can also be mediated by activation of other components of the immune system through cytokine release by CD4+ T cells. Long-term eradication and prevention of tumor recurrence can be provided by either immediate tumor resection or by long-term memory CAR-T cells that remain after the initial tumor response. Therefore, having CAR-T cells that are of the T stem cell memory phenotype (Tscm) and T central memory phenotype (Tcm) can be particularly advantageous. In addition, the release of non-BMCA tumor-associated antigens during CAR-T mediated tumor cell lysis can lead to a phenomenon called "epitope expansion," which is the priming and / or reactivation of unmanipulated tumor-specific cells in the unmanipulated adaptive immune system, potentially promoting long-term eradication and prevention of tumor recurrence.

[0376] In a non-clinical study conducted by Carpenter et al. (Carpenter, 2013), anti-BCMA-CAR T cells exhibited specific anti-BCMA functions, including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. Importantly, anti-BCMA-CAR T cells recognized and killed primary MM cells. The authors concluded that BCMA is a suitable target for CAR-expressing T cells, and that adoptive transfer of anti-BCMA-CAR T cells represents a promising novel strategy for treating MM (Carpenter, 2013).

[0377] Data from a trial conducted at the National Cancer Institute (NCI) have been published that provide the first clinical proof of the concept of T cells expressing anti-BCMA CARs in patients with relapsing / refractory MM. Patients (N=16) were enrolled at four dose levels, with cells measured at 0.3 × 10⁶ 6 , 1 x 10 6 , 3 x 10 6 , and 9×10 6The data included cells / kg body weight. Responses were dose-dependent and included partial responses (N=3), very good partial responses (N=4), and exact complete responses (N=1). At the highest dose level, the response rate was 100%. Data on the duration of response are not yet available. Tolerability was consistent with predictions from other CAR-T trials. Toxicity attributable to anti-BCMA CAR-T cells was minimal in patients treated at low dose levels. Subsequently, patients showed signs of CRS correlated with dose level. No unexpected damage to non-hematopoietic organs was observed (Ali, 2016, Kochenderfer, 2016). Initial results from another trial using anti-BCMA CAR-T cells by Cohen et al. (Cohen, 2016) have recently been published. In this trial, six patients were treated with 1–5 × 10⁸ CAR-T cells. Four patients demonstrated a response, including minimal response (N=2), very good partial response (N=1), and exact complete response with minimal residual disease (MRD) negativity (N=1). Five patients developed CRS, one of which was grade 3, and showed neurotoxicity that responded to treatment without sequelae. CAR-T cell proliferation appeared to correlate with efficacy. In an ongoing trial of a third anti-BCMA CAR-T construct published by Berdeja et al., 11 patients were enrolled at three fixed-dose levels, with 5 × 10⁶ CAR-T+ cells per patient. 7 pieces, 15×10 7 pieces, and 45 × 10 7 It was one (80 x 10 CAR-T+ cells per patient) 7 individual and 120 × 10 7(The number of individuals will be planned for the next cohort.) The response rate was impressive and dose-dependent, including partial response (N=4), very good partial response (N=1), and exact complete response (N=2), with two patients becoming MRD-negative. Tolerability was better than that reported in other CAR-T trials. CRS was observed in 70–80% of patients but was limited to grade 1–2. No neurotoxicities or other serious or unexpected toxicities were reported (Berdeja, 2016). The relatively low toxicity in these trials has been attributed to various factors, including the use of the 4-1BB costimulatory domain, reduced disease burden, and / or more graded exposure of T cells to myeloma cells, compared to the anti-CD19 products used in leukemia that have produced the most publications.

[0378] Lenalidomide (2019) is a member of the immunomodulatory imide (IMiD) class, known for its remarkable activity against myeloma and its multifaceted effects on the immune system. It is approved in the United States for the treatment of myeloma, myelodysplastic syndromes, and lymphoma (Moreau, 2019, Fink, 2015). In addition to its direct anti-myeloma properties, it has been hypothesized that lenalidomide can enhance the efficacy of CAR-T cells such as P-BCMA-101. Lenalidomide treatment increases the frequency of naive and stem cell memory T cells, and these two T subsets are not only preferred starting materials for the P-BCMA-101 manufacturing process but are also associated with improved clinical outcomes in CAR-T cell products (Fostier, 2018, Barnett, 2016a, Cohen, 2019). Direct inclusion of lenalidomide in the manufacturing process has not been proposed because this approach may increase the number and effector function of the CAR-T cells produced (Wang, 2018). Therefore, it is hypothesized that including lenalidomide treatment before apheresis improves the quality of T cells in the input material and subsequently improves the manufactured CAR-T cell product. Co-administration of lenalidomide and CAR-T cells has been proposed because it has been shown to enhance the effector function and overall anti-myeloma activity of CAR-T cells in both in vitro and mouse models (Otahal, 2016, Wang, 2018, Works, 2019). Furthermore, a similar combination of lenalidomide and anti-BCMA CAR-T cell products is currently being explored in at least one other clinical trial (NCT03070327).

[0379] Rituximab (2019) is an anti-CD20 antibody therapy that depletes CD20+ cells, i.e., B cells, and has a superior safety and efficacy profile. It is approved in the United States for the treatment of numerous lymphomas, leukemias, and autoimmune diseases (Salles, 2017). A therapeutic synergy is expected between P-BCMA-101 and rituximab for several reasons. CD20 expression has been well demonstrated in a subset of multiple myeloma, possibly including multiple myeloma stem cells (Flores-Montero, 2016, Matsui, 2018, Kapoor, 2008). While there is some debate as to whether multiple myeloma stem cells uniformly express CD20, there is a consensus that myeloma differentiation parallels the development of normal B cells, in which BCMA-CD20+ cells eventually mature into BCMA+CD20- cells (Johnsen, 2016, Bodker, 2018). A combined strategy of targeting BCMA+ cells with CAR-T cells and CD20+ cells with rituximab was able to eradicate mature neoplasms and any pre-malignant cells that could cause relapse. Furthermore, lymphopenia is known to contribute to the efficacy of CAR-T cell therapy, and rituximab has been shown to prolong this state (Cohen, 2019; Yutaka, 2015). Finally, anti-CAR antibodies have been observed in patients with multiple myeloma treated with CAR-T cells, which may limit product durability and suggest that clinical response may be improved by depletion of the endogenous B cell compartment (Xu, 2019).

[0380] 1.2.3. Design and Rationale of P-BCMA-101 P-BCMA-101 cells are designed to express three key components: the anti-BCMA centintine chimeric antigen receptor (CARTyrin) gene, the dihydrofolate reductase (DHFR) resistance gene, and the inducible caspase 9 (iC9)-based safety switch gene (Hermanson, 2016). BCMA-specific CARTyrin binding of the BCMA antigen expressed on the surface of MM tumor cells triggers intracellular signaling and activation in P-BCMA-101 cells, mediated by the cytoplasmic signaling domain encoded by CARTyrin. Beyond the intrinsic anti-BCMA moiety encoded by CARTyrin, the main difference between the P-BCMA-101 approach and most other CAR-T cells lies in the manufacturing process.

[0381] While the genetic modification of autologous T cells for CAR molecule expression is generally performed by lentiviral or γ-retroviral transduction, P-BCMA-101 is manufactured using an electroporation-based non-viral (DNA transposon) system called the piggyBac (PB) DNA modification system (Nakazawa, 2013), which efficiently transfers DNA from plasmids to chromosomes via a "cut and paste" mechanism and has been widely used as a human gene transfer method, including CAR-T production (Woodard, 2015, Fraser, 1996, Singh, 2013, Huls, 2013). Compared to virus-based delivery, PB offers advantages including a safer insertion profile (Cunningham, 2015), larger transgene capacity (enabling delivery of genetic components that enhance safety and efficacy), higher levels and more stable transgene expression (Cunningham, 2015), longer duration of transgene expression (Mossine, 2013), and the superiority of a highly favorable TSCM phenotype. In the case of P-BCMA-101, Super piggyBac transposase (SPB) is used, which is an engineered, highly reactive enzyme that catalyzes the integration of PB transposons into TTAA sites in the target genome, resulting in a clearer and safer integration profile. While the gene cargo capacity of lentiviruses and γ-retroviruses is limited to approximately 10-20 Kb, the piggyBac DNA modification system has demonstrated a cargo limit of >300 Kb, enabling the introduction of multiple beneficial genes. Furthermore, compared to lentiviruses, PB-mediated transduction allows for high levels of sustained expression of the tra...

Claims

1. A first composition for use in the treatment of cancer, in combination with a second composition containing an anti-CD20 agent, which contains a population of T cells expressing a chimeric antigen receptor (CAR), The CAR includes an antigen recognition domain that specifically binds to B cell maturation antigen (BCMA), The aforementioned cancer includes BCMA+ tumor cells, The aforementioned anti-CD20 agent is rituximab, ofatumumab, ocrelizumab, iodine i131 tositumomab, obinutuzumab, or ibritumomab. A first composition for the aforementioned use.

2. The first composition for use according to claim 1, wherein the anti-drug antibody (ADA) response to the first composition in the patient is reduced by at least 50% compared to a patient who is administered the first composition but not the second composition.

3. The first composition for use according to claim 1, wherein the persistence of the first composition in the patient is increased by at least 75%, and optionally at least 90%, compared to a patient who is administered the first composition but not the second composition.

4. The first composition for use according to claim 3, wherein the measure of persistence is the area under the plasma concentration curve (AUC).

5. A first composition for use according to any one of claims 1 to 4, further comprising a third composition comprising at least one lymphocyte depletion agent.

6. The first composition for use according to any one of claims 1 to 5, wherein the anti-CD20 agent is rituximab.

7. The first composition for use according to any one of claims 1 to 6, wherein the antigen recognition domain comprises centintin, scFv, a single-domain antibody, VH, or VHH.

8. The first composition for use according to any one of claims 1 to 7, wherein the antigen recognition domain comprises centintin.

9. The first composition for use according to any one of claims 1 to 8, wherein the first composition is CARTYrin.

10. The aforementioned CARTyrin is as follows: (a) Human CD8a signal peptide, (b) Centilin that specifically binds to BCMA, (c) Human CD8a hinge region, (d) Human CD8a transmembrane region, (e) Human 4-1BB co-stimulatory domain, and (f) CD3ζ costimulatory domain, A first composition for use according to claim 9, comprising:

11. The following features: (a) The human CD8a signal peptide comprises the amino acid sequence of SEQ ID NO: 3, (b) The centintrinity that specifically binds to BCMA includes the amino acid sequence of SEQ ID NO: 41, (c) The human CD8a hinge region comprises the amino acid sequence of Sequence ID No. 10, (d) The human CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO: 4, (e) The human 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 8, and (f) The CD3ζ co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 6, A first composition for use according to claim 10, comprising:

12. The first composition for use according to any one of claims 9 to 11, wherein the CARTyrin comprises the amino acid sequence of SEQ ID NO:

42.

13. The first composition is administered as multiple injections, and the multiple injections comprise a total dose divided into a first injection and a second injection. i) The first injection comprises about one-third of the total volume, ii) The first composition for use according to any one of claims 1 to 12, wherein the second infusion comprises about two-thirds of the total dose and is administered at least 10 days after the first infusion.

14. The first composition is administered as multiple infusions, the multiple infusions comprising a total dose divided into a first infusion, a second infusion, and a third infusion. i) The first injection comprises about one-third of the total volume, ii) The second infusion comprises about one-third of the total dose and is administered at least 10 days after the first infusion. iii) The first composition for use according to any one of claims 1 to 12, wherein the third infusion comprises about one-third of the total dose and is administered at least 10 days after the second infusion.

15. The first composition for use according to claim 13 or 14, wherein the time between the first injection and the second injection, or the time between the second injection and the third injection, is at least one week, two weeks, three weeks, four weeks, five weeks, or six weeks.

16. A first composition for use according to any one of claims 1 to 15, wherein the first composition, the second composition, and / or the third composition are administered sequentially or simultaneously.

17. The first composition for use according to any one of claims 5 to 16, wherein the third composition is administered before the first composition.

18. The third composition is administered once daily, and a first dose of the third composition is administered at least five days prior to the first infusion of the first composition. The first composition for use according to claim 17, wherein the third composition is optionally administered three, four, and five days before the first infusion of the first composition.

19. The first composition for use according to any one of claims 1 to 18, wherein the second composition is administered before the first composition.

20. A first composition for use according to any one of claims 1 to 19, wherein the second composition or the third composition is administered in two or more doses.

21. The first composition for use according to claim 20, wherein a first dose of the second composition is administered 12 days before the first infusion of the first composition, a second dose of the second composition is administered 5 days before the first infusion of the first composition, and subsequent doses are administered once a week for at least 8 weeks after the first infusion of the first composition.

22. The first composition for use according to any one of claims 1 to 21, wherein the subject has not been previously treated with an anticancer drug.

23. The first composition for use according to any one of claims 5 to 22, wherein the first lymphocyte depletion agent of the third composition and the second lymphocyte depletion agent of the third composition are administered simultaneously or sequentially.

24. The first composition for use according to claim 23, wherein the first lymphocyte depletion agent and the second lymphocyte depletion agent are administered on the same day, the first lymphocyte depletion agent is administered intravenously over a period of 30 minutes, and the second lymphocyte depletion agent is administered intravenously over a period of 30 minutes.

25. The first composition for use according to claim 23 or 24, wherein the first lymphocyte depletion agent or the second lymphocyte depletion agent is cyclophosphamide or fludarabine.

26. The dose of the third composition is i) 100 mg / m² ２ , 200 mg / m² ２ , 300 mg / m² ２ , 400 mg / m² ２ , or 500 mg / m² ２ Cyclophosphamide, ii) 10 mg / m ２ , 20 mg / m ２ , 30 mg / m ２ , 40 mg / m ２ , or 50 mg / m ２ of fludarabine, or a combination thereof, Optionally, the dose of the third composition may be 300 mg / m². ２ Cyclophosphamide and 30 mg / m² ２ A first composition for use according to claim 23, comprising fludarabine.

27. The first composition contains at least 0.1 × 10 cells ６ , 0.2 × 10 ６ , 0.25 × 10 ６ , 0.5 × 10 ６ , 0.6 × 10 ６ , 0.7 × 10 ６ , 0.75 × 10 ６ , 0.8 × 10 ６ , 0.9 × 10 ６ , 1 x 10 ６ , 2 x 10 ６ , 3 x 10 ６ , 4 x 10 ６ , 5 x 10 ６ , 6 x 10 ６ , 7 x 10 ６ , 8 x 10 ６ , 9 x 10 ６ , 10 x 10 ６ , 11 x 10 ６ , 12 x 10 ６ , 13 x 10 ６ , 14 x 10 ６ , 15 x 10 ６ , 16 x 10 ６ , 17 x 10 ６ , 18 x 10 ６ , 19 x 10 ６ , or 20 x 10 ６ A first composition for use according to any one of claims 1 to 26, administered in a total dose of units / kg of the body weight of the subject.

28. The first injection, the second injection, and / or the third injection of the first composition delivers the first composition to approximately 1 × 10 cells. ５ pieces / mL ~ approx. 5 x 10 ７ It is administered using an infusion bag containing the particles / mL at a concentration of , Optionally, the infusion bag may contain the first composition in approximately 3 × 10 cells. ５ pieces / mL ~ approx. 2.4 x 10 ７ Contains at a concentration of 1 / mL A first composition for use according to any one of claims 13 to 27.

29. The dose of the second composition is 100 mg / m². ２ , 125 mg / m² ２ , 150 mg / m² ２ , 175 mg / m² ２ , 200 mg / m² ２ , 225 mg / m² ２ , 275 mg / m² ２ , 300 mg / m² ２ , 325 mg / m² ２ , 375 mg / m² ２ , 400 mg / m² ２ , 425 mg / m² ２ , 450 mg / m² ２ , 475 mg / m² ２ , or 500 mg / m² ２ Contains rituximab, Optionally, the dose of the second composition may be 375 mg / m². ２ It is rituximab, A first composition for use according to any one of claims 1 to 28.

30. The first composition for use according to claim 29, wherein the second composition is administered by intravenous infusion, and the flow rate of the intravenous infusion is approximately 25 mg / hour to approximately 500 mg / hour.

31. The first composition for use according to claim 30, wherein the first dose of the second composition is administered by intravenous infusion at a flow rate of 50 mg / hour, the flow rate being increased every 30 minutes up to a maximum of 400 mg / hour.

32. The first composition for use according to claim 31, wherein the second dose and subsequent doses of the second composition are administered by intravenous infusion at a flow rate of about 100 mg / hour, with the flow rate increasing every 30 minutes up to a maximum of about 400 mg / hour.

33. The first composition for use according to any one of claims 1 to 32, wherein the cancer is multiple myeloma.

34. The first composition for use according to claim 33, wherein the multiple myeloma is relapsed multiple myeloma or refractory multiple myeloma.

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