CAR-T cell therapy targeting BCMA in multiple myeloma

Abstract

This provides a method for treating individuals with cancer. [Solution] At least a single dose of chimeric antigen receptor (CAR)-T cells containing a polypeptide-containing CAR is administered to the subject. The peptide comprises an extracellular antigen-binding domain having at least two BCMA-binding moieties, a transmembrane domain, and an intracellular signaling domain. The dose of CAR-T cells administered to the subject is 4.0 × 10⁶ per kilogram of the subject's mass. 5 ~1.0×10 6 These are CAR-T cells. Alternatively, the dose is 1 × 10⁶ 6 ~1 × 10 8 Contains 1 CAR-T cell.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is a U.S. Provisional Application No. 62 / 931,065 (filed November 5, 2019) and the same Claiming priority to Patent No. 62 / 943,627 (filed December 4, 2019), Both of them are incorporated herein by reference in their entirety.

[0002] (Sequence Listing) This application has been submitted electronically in ASCII format, and the entire application is by reference. This specification includes sequence listings incorporated herein. The above ASCII copy is valid until October 30, 2020. Created on [date], named 253505_000091_SL.txt, size 9, It is 516 bytes. [Background technology]

[0003] Multiple myeloma is an invasive neoplasm of plasma cells. It is thought to be a B-cell neoplasm that proliferates uncontrollably. Symptoms include high calcium levels. One of the following conditions: blood smegma, renal failure, anemia, bone lesions, bacterial infection, hyperviscosity, and amyloidosis. One or more of the following are examples of proteasome inhibitors with significantly improved patient outcomes. Despite new therapies including immunomodulatory drugs and monoclonal antibodies, multiple myeloma It remains an incurable disease. [Overview of the project] [Problems that the invention aims to solve]

[0004] Most patients experience relapses or become refractory to treatment, thus multiple myeloma There is a continuing need for new therapies for this purpose. [Means for solving the problem]

[0005] This specification provides improved therapies for treating multiple myeloma.

[0006] In one embodiment, a method for treating a subject with cancer is provided, and this method is a) Extracellular antigen-binding dormant containing a first anti-BCMA binding moiety and a second BCMA binding moiety In and b) Transmembrane domain and, c) A chimeric antigen receiver containing an intracellular signaling domain. To administer at least a single dose to cells containing eptor (CAR) polypeptides. include.

[0007] In some embodiments, cells are grown in vitro before injection. Morphologically, the cells are T cells, NK cells, and iPSC-NK cells. Several embodiments So, the cells are CAR-T cells. In some embodiments, the cells are NKT cells. These are iPSC-T cells, or gamma-delta T cells. In some embodiments, the cells are , different species or oneself.

[0008] In some embodiments, the dose is 4.0 × 10⁻¹⁶ per kilogram of the mass of the subject. 5 ~ 1.0 × 10 6 Contains 1 CAR-T cell. In some embodiments, the dose is of the quality of the subject. 4.0 × 10 per kilogram 5 ~1.0×10 6 Individual viable CAR-T cells Includes. In some embodiments, the dose is 4.0 × 1 per kilogram of the mass of the subject. 0 5 ~6.0×10 5comprises individual CAR-T cells. In some embodiments, the dosage is 5.0×10 to 7.0×10 5 individual CAR-T cells per kilogram of the subject's mass 5 . In some embodiments, the dosage is 5.5×10 to 6.0×10 5 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the dosage is 5 6.0×10 to 8.0×10 5 individual CAR-T cells per kilogram of the subject's mass 5 . In some embodiments, the dosage is 7.0×10 5 to 9 .0×10 5 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the dosage is 8.0×10 5 to 1.0×10 6 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the dosage is about 5.0×10 5 , about 6 .0×10 5 , about 7.0×10 5 , about 8.0×10 5 , about 9.0×10 5 , or about 1.0 ×10 6 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the dosage is about 7.1×10 5 , about 7.2×10 5 , about 7.3×10 5 , about 7.4× 10 5 , about 7.5×10 6 , about 7.6×10 6 , about 7.7×10 6 , about 7.8×10 6 , or about 7.9×10 6Contains 1 CAR-T cell. In one embodiment, the dose is the mass of the subject. Approximately 7.5 x 10 per kilogram 5 Contains 1 CAR-T cell.

[0009] In some embodiments, the dose is 1 × 10 6 ~1 × 10 8 Contains individual CAR-T cells In some embodiments, the dose is 1 × 10 6 ~5×10 6 Contains individual CAR-T cells In some embodiments, the dose is 410 6× ~1.5×10 7 individual CAR-T cells Includes. In some embodiments, the dose is 1 × 10 7 ~2.5×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 2 × 10 7 ~4×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 3 × 10 7 ~5×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 410 7× ~6×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 5 × 10 7 ~7×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 6 × 10 7 ~8×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 710 7× ~9×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is 8 × 10 7 ~1 × 10 8 Individual CAR-T details Contains cells. In some embodiments, the dose is 2 × 107 ~8×10 7 Individual CAR-T details Contains cells. In some embodiments, the dose is about 1 × 10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9× 10 7 , or approximately 1 x 10 8 Contains 1 CAR-T cell. In some embodiments, the dose is , about 5.1×10 7 , about 5.2×10 7 , about 5.25×10 7 , about 5.3×10 7 , about 5 .4×10 7 , about 5.5×10 7 , about 5.6×10 7 , about 5.7×10 7 , about 5.8×1 0 7 , or approximately 5.9 × 10 7 Contains CAR-T cells. In one embodiment, the dose is approximately 5 .25×10 7 Contains 1 CAR-T cell.

[0010] In some embodiments, the cells are 4.0 × 10⁶ per kilogram of the mass of the subject. 5 ~ 1.0 × 10 6 It is administered in a dose that yields a certain number of CAR-T cells. In some embodiments The cells are 4.0 × 10⁶ per kilogram of mass of the subject. 5 ~1.0×10 6 Individual survival It is administered in a dose that yields possible CAR-T cells. In some embodiments, the cells are 4.0 × 10⁻¹⁰ per kilogram of the mass of the object 5 ~6.0×10 5administered at a dose that provides the individual CAR-T cells. In some embodiments, the cells are administered at a dose that provides 5.0×10 5 ~7.0×10 5 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the cells are administered at a dose that provides 5.5×10 5 ~6.0×10 5 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the cells are administered at a dose that provides 6.0×10 5 ~8.0×10 5 individual C AR-T cells per kilogram of the subject's mass. In some embodiments, the cells are administered at a dose that provides 7.0×10 5 ~9.0×10 5 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the cells are administered at a dose that provides 8.0×10 5 ~1.0×10 6 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the cells are administered at a dose that provides about 5.0×10 5 , about 6 .0×10 5 , about 7.0×10 5 , about 8.0×10 5 , about 9.0×10 5 , or about 1.0 ×10 6 individual CAR-T cells per kilogram of the subject's mass. In some embodiments, the cells are administered at a dose that provides about 7.1×10 5 , about 7.2×10 5 , about 7.3 ×10 5 , about 7.4×10 5 , about 7.5×10 6 , about 7.6×106 , about 7.7×10 6 , about 7.8×10 6 , or approximately 7.9 × 10 6 Administered in a dose that yields individual CAR-T cells. In one embodiment, the cells are approximately 7.5 × 10¹⁶ per kilogram of the mass of the subject. 5 individual It is administered in a dose that induces CAR-T cells.

[0011] In some embodiments, the cells are 1 × 10 in the subject. 6 ~1 × 10 8 Individual CAR- It is administered in a dose that produces T cells. In some embodiments, the cells are 1 in the subject ×10 6 ~5×10 6 It is administered in a dose that yields a certain number of CAR-T cells. Several implementations Morphologically, the cells are 4 × 10 in the subject. 6 ~1.5×10 7 Having individual CAR-T cells It is administered in a dose of 1 × 10⁶. In some embodiments, the cells are 1 × 10⁶ in the subject. 7 ~2 0.5×10 7 It is administered in a dose that yields a certain number of CAR-T cells. In some embodiments, The cells were 2 × 10 in the subject. 7 ~4×10 7 In a dose that produces individual CAR-T cells, administer In some embodiments, the cells are given 3 × 10 in the subject. 7 ~5×10 7 individual It is administered in a dose that yields CAR-T cells. In some embodiments, the cells are administered to the target. 4 x 10 7 ~6×10 7 It is administered in a dose that yields a certain number of CAR-T cells. In one embodiment, the cells are 5 × 10 in the subject. 7 ~7×10 7individual CAR-T cells It is administered in a dose that yields the desired result. In some embodiments, the cells are 6 × 10 in the subject. 7 ~8×10 7 It is administered in a dose that yields a certain number of CAR-T cells. In some embodiments, The cells were 7 × 10 in the subject. 7 ~9×10 7 In a dose that produces individual CAR-T cells, administer In some embodiments, the cells are given 8 × 10 in the subject. 7 ~1 × 10 8 individual It is administered in a dose that yields CAR-T cells. In some embodiments, the cells are administered to the target. 2 × 10 7 ~8×10 7 It is administered in a dose that yields a certain number of CAR-T cells. In one embodiment, the cells are approximately 1 × 10⁶ in the subject. 7 , about 2×10 7 , about 3×10 7 , Approximately 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9×10 7 , or approximately 1 x 10 8 It is administered in a dose that yields a certain number of CAR-T cells. Several implementations Morphologically, the cells are approximately 5.1 × 10⁻⁶ in the subject. 7 , about 5.2×10 7 , about 5.25×1 0 7 , about 5.3×10 7 , about 5.4×10 7 , about 5.5×10 7 , about 5.6×10 7 ,about 5.7 × 10 7 , about 5.8×10 7 , or approximately 5.9 × 10 7 It gives a number of CAR-T cells It is administered in a dose. In one embodiment, the cells are approximately 5.25 × 10 in the subject. 7 Individual C It is administered in a dose that produces AR-T cells.

[0012] In some embodiments, the dose of CAR-T cells is administered only once during the course of treatment. In some embodiments, the dose of CAR-T cells is administered intravenously. In this embodiment, the cancer is multiple myeloma. In a specific embodiment, multiple myeloma is intractable. It is either recurrent multiple myeloma or relapsed multiple myeloma.

[0013] In various embodiments, the CD8 of CD4+ CAR-T cells in CAR-T cell doses The ratio to +CAR-T cells is less than 4. In some embodiments, CAR-T cells The ratio of CD4+ CAR-T cells to CD8+ CAR-T cells at the dose of cells is 2. It is full. In some embodiments, the dose of CAR-T cells is CD4+CAR-T The ratio of cells to CD8+CAR-T cells is less than 1.8 in some embodiments. This relates to the difference between CD4+ CAR-T cells and CD8+ CAR-T cells in terms of CAR-T cell dose. The ratio is less than 1.7. In some embodiments, the dose of CAR-T cells The ratio of CD4+ CAR-T cells to CD8+ CAR-T cells is less than 1.6. In some embodiments, the CD4+ CAR-T cell dose The ratio to 8+CAR-T cells is less than 1.4. In some embodiments, CAR - The ratio of CD4+CAR-T cells to CD8+CAR-T cells at T cell dose is , less than 1.2. In some embodiments, the CD4+ dose of CAR-T cells The ratio of CAR-T cells to CD8+CAR-T cells is less than 1.0.

[0014] In some embodiments, effector memory CAR+ T cells are CAR-T cells The dose constitutes at least 20% of the total number of CAR+ T cells. Several embodiments So, effector memory CAR+ T cells are CAR+ at the dose of CAR- T cells. It constitutes at least 25% of the total T cell population. In some embodiments, effector Molly CAR+ T cells are less of a total amount of CAR+ T cells than CAR-T cells in terms of dose. They each make up 30%. In some embodiments, effector memory CAR+ T cells This constitutes at least 35% of the total amount of CAR+ T cells in terms of CAR-T cell dose. In some embodiments, effector memory CAR+ T cells are CAR-T cells. The dose constitutes at least 40% of the total number of CAR+ T cells. Several embodiments So, central memory CAR+ T cells are CAR+ T cells at the dose of CAR-T cells. It constitutes at least 3% of the total cell volume. In some embodiments, it is central memory. CAR+ T cells are at least 5 times the total amount of CAR+ T cells in the dose of CAR+ T cells. It constitutes %. In some embodiments, central memory CAR+ T cells are CAR -Constitutes at least 6% of the total amount of CAR+ T cells in terms of T cell dose. In this embodiment, central memory CAR+ T cells are C at the dose of CAR-T cells. It constitutes at least 10% of the total amount of AR+T cells. In some embodiments, the central Memory CAR+ T cells are small in the total amount of CAR+ T cells at the dose of CAR-T cells. It still makes up at least 15%.

[0015] CD4+ CAR-T cells at Cmax vs. CD8+ CAR-T cells at Cmax Various embodiments of the above method, where the ratio is less than 3.5. In some embodiments, Cma The ratio of CD4+ CAR-T cells to CD8+ CAR-T cells at Cmax in x is 2 It is less than 0.0. In some embodiments, the Cmax of CD4+ CAR-T cells is less than 0.0. The ratio to CD8+CAR-T cells in ax is less than 1.2. (Several embodiments) So, regarding CD4+ CAR-T cells at Cmax and CD8+ CAR-T cells at Cmax... The ratio is less than 0.8. In some embodiments, CD4+CAR- at Cmax The ratio of T cells to CD8+CAR-T cells at Cmax is less than 0.6. In that embodiment, CD4+CAR-T cells at Cmax and CD8+CAR-T cells at Cmax - The ratio to T cells is less than 0.4. In some embodiments, CD at Cmax The ratio of 4+CAR-T cells to CD8+CAR-T cells at Cmax is less than 0.3. be.

[0016] In the various embodiments described above, this method involves CD4+CAR-T cells and / or This further includes assaying the quantity of CD8+ CAR-T cells.

[0017] In various embodiments of the above method, central memory CAR+ T cells are administered in doses. Afterward, it constitutes at least 75% of the total amount of CAR+ T cells. In some embodiments, Central memory CAR+ T cells, after dose administration, the total amount of CAR+ T cells It constitutes at least 80% of the central memory CAR+. In some embodiments, the central memory CAR+ T cells constitute at least 85% of the total number of CAR+ T cells after dose administration. In some embodiments, central memory CAR+ T cells are, after dose administration, They constitute at least 90% of the total number of CAR+ T cells.

[0018] In various embodiments of the above method, the method involves administering a dose to the subject after the dose has been administered. Assay the ratio of total memory CAR+ T cells to the total amount of CAR+ T cells. This further includes the following.

[0019] In various embodiments of the above method, effector memory CAR+ T cells are administered in doses. After administration, it constitutes at least 2% of the total amount of CAR+ T cells. In some embodiments, Effector memory CAR+ T cells, after dose administration, total CAR+ T cells It constitutes at least 3% of the amount. In some embodiments, the central memory CAR+ T cells constitute at least 5% of the total CAR+ T cell population after dose administration. In some embodiments, central memory CAR+ T cells are C after dose administration. They constitute at least 8% of the total AR+ T cell population.

[0020] In various embodiments of the above method, the method involves administering a dose and then inf in the subject. Assay on the ratio of CAR+ T cells to the total amount of CAR+ T cells. This further includes the following.

[0021] In various embodiments of the above method, central memory CAR+CD8+ T cells are used After the dose is administered, at least the total amount of CAR+CD8+ T cells in the subject reaches Cmax. It constitutes 30%. In some embodiments, the central memory CAR+CD8+T is fine. The cells showed a decrease in the total amount of CAR+CD8+ T cells in the subjects at Cmax after dose administration. It constitutes at least 50%. In some embodiments, the central memory is CAR+CD. 8+ T cells were compared to CAR+CD8+ T cells in the control at Cmax after dose administration. It constitutes at least 70% of the total amount. In some embodiments, the central memory CA R+CD8+ T cells constitute at least 80% of the total number of CAR+CD8+ T cells.

[0022] In some embodiments, the method involves administering a dose and then measuring the size of the target at Cmax. The ratio of central memory CAR+CD8+ T cells to the total amount of CAR+CD8+ T cells The procedure further includes steps for performing an assay.

[0023] In some embodiments, effector memory CAR+CD8+ T cells are CAR+ It constitutes at least 2% of the total number of CD8+ T cells. In some embodiments, the effect Ter memory CAR+CD8+ T cells are at least 5% of the total amount of CAR+CD8+ T cells. It constitutes %. In some embodiments, effector memory CAR+CD8+T cells This constitutes at least 8% of the total amount of CAR+CD8+ T cells. In some embodiments, The effector memory CAR+CD8+T cells are the total amount of CAR+CD8+T cells It constitutes at least 10%.

[0024] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. The total amount of CAR+CD8+T cells in the effector memory The procedure further includes a step of assaying the ratio to the given value.

[0025] In various embodiments of the above method, central memory CAR+CD4+ T cells are used After the dose is administered, at least the total amount of CAR+CD4+ T cells in the subject reaches Cmax. It constitutes 5%. In some embodiments, central memory CAR+CD4+ T cells This is because, after dose administration, the total amount of CAR+CD4+ T cells in the subject is low at Cmax. It constitutes at least 8%. In some embodiments, the central memory CAR+CD4+ T cells, after dose administration, represent the total amount of CAR+CD4+ T cells in the subject at Cmax. It constitutes at least 10% of the central memory CAR+. In some embodiments, the central memory CAR+ CD4+ T cells were increased in the CAR+CD4+ T cell population at Cmax after dose administration. It constitutes at least 15% of the total volume of cells.

[0026] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. In relation to the total amount of CAR+CD4+T cells in central memory, The further step includes assaying with respect to the ratio.

[0027] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. The total amount of CAR+CD4+T cells in the effector memory The process further includes an assay step regarding the ratio, where the effector memory CA R+CD4+ T cells constitute at least 70% of the total number of CAR+CD4+ T cells. In some embodiments, effector memory CAR+CD4+ T cells are CAR+C It constitutes at least 75% of the total number of D4+ T cells. In some embodiments, the effect Ter memory CAR+CD4+ T cells are at least 8 of the total number of CAR+CD4+ T cells. It constitutes 0%. In some embodiments, the effector memory CAR+CD4+T is fine Cells constitute at least 90% of the total number of CAR+CD4+ T cells. In certain embodiments, The effector memory CAR+CD4+T cells are the total amount of CAR+CD8+T cells. 70-80%, 70-85%, 71-86%, 72-87%, 73-88%, 74-89 %, 75-90%, 76-91%, 77-92%, 78-93%, 80-90%, 82- It consists of 92%, 84-94%, 86-96%, 88-98%, or 90-100%.

[0028] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. The total amount of CAR+CD4+T cells in the effector memory The procedure further includes a step of assaying the ratio to the given value.

[0029] In some embodiments, a first BCMA binding portion and / or a second BCMA binding portion This is an anti-BCMA sdAb. In some embodiments, the first BCMA binding portion is The first anti-BCMA sdAb is, and the second BCMA binding site is the second anti-BCMA It is sdAb. In a particular embodiment, the first BCMA binding portion is QVKLEESGG GLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVA VIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPE DTAVYYCAARRIDAADFDSWGQGTQVTVSS (Sequence No. 1) It contains an acid sequence. In certain embodiments, the first BCMA binding moiety is CAGGTCAAA CTGGAAGAATCTGGCGGAGGCCTGGTGCAGGCAGGACGGA GCCTGCGCCTGAGCTGCGCAGCATCCGAGCACACCTTCAG CTCCCACGTGATGGGCTGGTTTCGGCAGGCCCCAGGCAAG GAGAGAGAGAGCGTGGCCGTGATCGGCTGGAGGGACATCT CCACATCTTACGCCGATTCCGTGAAGGGCCCGGTTCACCAT CAGCCGGGACAACGCCAAGAAGACACTGTATCTGCAGATG AACAGCCTGAAGCCGAGGACACCGCCGTGTACTATTGCG CAGCAAGGAGAATCGACGCAGCAGACTTTGATTCCTGGGGG Nucleic acid sequence CCAGGGCACCCAGGTGACAGTGTCTAGC (Sequence ID 2) Includes a polypeptide coded by.

[0030] In a particular embodiment, the second BCMA binding portion is EVQLVESGGGLVQAGG SLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAY YAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAAD Contains the amino acid sequence RKSVMSIRPDYWGQGTQVTVSS (SEQ ID NO: 3). In a particular embodiment, the second BCMA binding portion is GAGGTGCAGCTGGTGGA GAGCGGAGGCGGCCTGGTGCAGGCCGGAGGCTCTCTGAGG CTGAGCTGTGCAGCATCCGGAAGAACCTTCACAATGGGCT GGTTTAGGCAGGCACCAGGAAAGGAGAGGGAGTTCGTGGC AGCAATCAGCCTGTCCCCTACCCTGGCCTACTATGCCGAG AGCGTGAAGGGCAGGTTTACCATCTCCCGCGATAACGCCA AGAATACAGTGGTGCTGCAGATGAACTCCCTGAAACCTGA GGACACAGCCCTGTACTATTGTGCCGCCGATCGGAAGAGC GTGATGAGCATTAGACCAGACTATTGGGGGCAGGGAACAC The nucleic acid sequence AGGTGACCGTGAGCAGC (SEQ ID NO: 4) encodes the port Contains lipeptides.

[0031] In some embodiments, the first BCMA binding portion and the second BCMA binding portion are ped They are connected to each other via a peptide linker. In certain embodiments, a peptide linker It contains the amino acid sequence GGGGS (SEQ ID NO: 5).

[0032] In some embodiments, the CAR polypeptide is located at the N-terminus of the polypeptide. It further contains signal peptides. In some embodiments, the signal peptide is CD8α It originates from. In certain embodiments, the signal peptide is MALPVTALLLPLALL Includes the amino acid sequence of LHAARP (SEQ ID NO: 6). In certain embodiments, the signal peptide Chido is ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTG The nucleic acid sequence GCTCTGCTGCTGCACGCTGCTCGCCCT (Sequence ID 7) Therefore, it includes the encoded polypeptide.

[0033] In certain embodiments, the transmembrane domain is IYIWAPLAGTCGVLLLSLVI Contains the amino acid sequence of TLYC (SEQ ID NO: 8).

[0034] The transmembrane domain is ATCTACATCTGGGCGCCCTTGGCCGGGACT TGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACT A specific implementation including a polypeptide encoded by the nucleic acid sequence GC (sequence number 9) Status.

[0035] In some embodiments, the intracellular signaling domain is the primary domain of immune effector cells. It contains essential intracellular signaling domains. The intracellular signaling domains are derived from CD3ζ Several embodiments result. In some embodiments, the intracellular signaling domain is , comprising one or more co-stimulatory signaling domains. In certain embodiments, intracellular The signaling domain is RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA Contains the amino acid sequence of LPPR (SEQ ID NO: 10).

[0036] In certain embodiments, the intracellular signaling domain is AGAGTGAAGTTCAG CAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTG TACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC Contains polypeptide encoded by the nucleic acid sequence GCTAA (SEQ ID NO: 11). In this embodiment, the intracellular signaling domain is KRGRKKLLYIFKQPFMR Amino acid sequence of PVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12) Includes. In certain embodiments, the intracellular signaling domain is AAACGGGGCAG AAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(Sequence ID) It contains a polypeptide encoded by the nucleic acid sequence 13).

[0037] CAR polypeptides have a C-terminus of the extracellular antigen-binding domain and an N-terminus of the transmembrane domain. Several embodiments further include hinge domains located between them. In certain embodiments, The hinge domain is TTTPAPRPPTPAPTIASQPLSLRPEACRPAA Contains the amino acid sequence GGAVHTRGLDFACD (SEQ ID NO: 15). Specific Embodiments So, the hinge domain is ACCACGACGCCAGCGCCGCGACCACCAA CACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC Nucleic acid sequence of ACGAGGGGGCTGGACTTCGCCTGTGAT (Sequence ID 14) Includes a polypeptide coded by.

[0038] In various embodiments, the T cells are autologous T cells. In some embodiments, T cells These are allogeneic T cells. In some embodiments, the subjects are human. [Brief explanation of the drawing]

[0039] [Figure 1] This shows the expression of BCMA antigens on the surface of germinal centers (GCs), memory cells, and plasmablasts in lymph nodes, long-lived plasma cells in bone marrow (LNs) and MALTs, and multiple myeloma cells. BAFF-R antigen is not expressed in plasmablasts, long-lived plasma cells, or multiple myeloma cells. TACI is expressed in memory cells, plasmablasts, long-lived plasma cells, and multiple myeloma cells. CD138 is expressed only on long-lived plasma cells and multiple myeloma cells. [Figure 2A] The design of the LCAR-B38M CAR is shown. In contrast to the single VL domain and single VH domain found in various other CARs, LCAR-B38M contains two VHH domains connected via a linker. LCAR-B38M contains intracellular CD137 and human CD3ζ domains. [Figure 2B] A schematic diagram is shown for preparing the LCAR-B38M CAR-encoding virus, transduction of the virus from a patient into T cells, and subsequent preparation of CAR T cells expressing LCAR-B38M. [Figure 3]This diagram shows a schematic of the MMY2001 study design for LCAR-B38M CAR T cells (LCAR-B38M). The patient population includes those with relapsed multiple myeloma, or refractory multiple myeloma with three prior treatment options to proteasome inhibitors (PIs) / immunomodulatory drugs (IMiDs) and prior PIs, IMiDs, αCD38 exposure, or dual refractory status. The primary objective is the safety and establishment of the recommended phase II dose (RP2D), including studies of the incidence and severity of adverse events (Phase 1b). Another primary objective is efficacy: overall response rate (ORR) - partial response (PR) or better, as defined by the International Myeloma Working Group (IMWG) (Phase 2). The following are the second objectives: assessment of adverse event incidence and severity (Phase 2), immunogenicity, post-treatment PRO and health-related quality of life (HRQoL), characterization of pharmacokinetics (PK) and pharmacodynamics (PD), and any further efficacy characterization. [Figure 4] Figure 3 summarizes the clinical responses in the MMY2001 study. Individual patient outcomes in the study are shown in a chart indicating treatment intervals and events. [Figure 5A] Summarize the minimal residual disease (MRD) status of study patients at days 28, 56, 184, and 365. Bold text indicates a negative MRD status, squared text indicates a positive MRD status, and "Uncertain" indicates an uncertain MRD status. NGS refers to ClonoSeq next-generation sequencing, and F refers to flow cytometry analysis. [Figure 5B]Summarize the minimal residual disease (MRD) status of study patients at days 28, 56, 184, and 365. Bold text indicates a negative MRD status, squared text indicates a positive MRD status, and "Uncertain" indicates an uncertain MRD status. NGS refers to ClonoSeq next-generation sequencing, and F refers to flow cytometry analysis. [Figure 6] This paper summarizes the translational research on CAR-T cell therapy in the MMY2001 study. [Figure 7] This paper summarizes the various biomarker evaluations conducted in the MMY2001 study. [Figure 8A] This graph shows the results of the qPCR assay for the number of transgene copies per microgram of gDNA for each patient in the MMY2001 study. Cmax and the time to the peak of proliferation (Tmax) can be seen in the graph. Cmax is highly variable among patients, while Tmax is consistent among patients. Empty dots indicate that the number of CAR+ T cells was below the lower limit of quantification (LLOQ) (5 cells / μl, range 2-7) in 11 out of 16 patients at least 8 weeks of follow-up. [Figure 8B] This graph shows the results of flow cytometry assays for the percentage of CD3 CAR+ T cells in total T cells for each patient in the MMY2001 study. Cmax and time to peak proliferation (Tmax) can be seen in the graph. While Cmax is highly variable among patients, Tmax is consistent across patients. Empty dots indicate that the number of CAR+ T cells was less than LLOQ (5 cells / μl, range 2–7) in 11 out of 16 patients at least 8 weeks of follow-up. [Figure 8C]This graph shows the results of flow cytometry assays for each patient in the MMY2001 study, indicating the number of CAR+CD3 T cells per microliter. Cmax and the time to the peak of proliferation (Tmax) can be seen in the graph. While Cmax is highly variable among patients, Tmax is consistent across patients. Empty dots indicate that in 11 of the 16 patients, the number of CAR+ T cells was less than LLOQ (5 cells / μl, range 2–7) at least 8 weeks of follow-up. [Figure 9A] The results of an assay demonstrating that bb2121CAR-T proliferation and persistence are dose-dependent are shown. Cells with 5.0 × 10⁷ cells did not persist beyond LLOQ after 2 months. Higher persistence was observed in cells with 1.50 × 10⁸ to 8.00 × 10⁸ cells. [Figure 9B] The results of the LCAR-B38M persistence assay in 5.25 × 10⁷ cells are shown, which corresponds to 0.75 × 10⁶ CAR-positive viable T cells / kg. The persistence is similar to that observed in bb2121 in 5.0 × 10⁷ cells in Figure 9A. [Figure 10A] This graph shows the persistence of indicators of CAR-T cell proliferation. The X-axis of the graph in Figure 10A represents the Cmax of the CAR+T cell assay as a percentage of T cells. [Figure 10B] This graph shows the persistence of indicators of CAR-T cell proliferation. The X-axis of the graph in Figure 10B represents the Cmax of the assay for transgene copies per microgram of gDNA. The degree of CAR-T cell proliferation does not predict persistence. [Figure 11A] This graph plots the Cmax (determined by the number of vector-transfected copies per microgram of genomic DNA) for responders and non-responders to bb2121CAR-T therapy. The relationship between Cmax and bb2121 response can be observed. [Figure 11B]An example graph plots the Cmax (determined by both qPCR and flow cytometry assays) of various classes of responders to LCAR-B38M CAR-T therapy (SD, PR, VGPR, CR, and sCR). Unlike bb2121, there is no correlation between clinical response and either Cmax or persistence. [Figure 11C] This graph plots double-positive (DP) CD4 / CD8 ratio against copies / microgram DNA. [Figure 11D] This graph plots the percentage of CD3 at Cmax against the DP CD4 / CD8 ratio. The DP CD4 / CD8 ratio does not correlate with growth. [Figure 12] The graph shows the percentage of CD3 at Cmax versus tumor volume. The results indicate that tumor volume does not correlate with LCAR-B38M proliferation (Cmax). [Figure 13A] The graph shows the concentration of CD3 cells per microliter versus tumor volume at Cmax. The results indicate that tumor volume does not correlate with LCAR-B38M proliferation (Cmax). [Figure 13B] The graph shows the plot of transgene copies versus tumor load per microgram of gDNA at Cmax. The results indicate that tumor load does not correlate with LCAR-B38M proliferation, as shown by Cmax. [Figure 14A] This graph shows the CD4:CD8 ratio (Y-axis) during peak CAR T-cell proliferation versus the CD4:CD8 ratio (X-axis) in the final bb2121 cell product. The final bb2121 CAR+ T-cell product consisted of a variable proportion of CAR+CD4 and CD8 T cells, with a median of 85% (range 42-98) for CAR+CD4+ T cells and 13% (range 2-47) for CAR+CD8+ T cells. The CD4:CD8 ratio in DP cells was 6.54. A correlation was observed between the CAR+CD4:CD8 T-cell ratio observed in the final product and during peak proliferation. [Figure 14B]This graph shows the CD4:CD8 ratio in peak CAR T-cell proliferation (Y-axis) versus the CD4:CD8 ratio in DP CART+ products (X-axis). The median CD4:CD8 ratio in DP cells is 1.54. The median CD4:CD8 ratio in cells at Cmax is 0.35. [Figure 15] This graph shows the CD4:CD8 ratio of LCAR-B38M in various patients from the MMY2001 study at specific days before and after infusion (day zero). In most patients, the CD4:CD8 ratio was less than 1 at peak proliferation. [Figure 16A] This shows that LCAR-B38M is enriched in CD8 CAR+ T cells at the peak of proliferation in the bone marrow. Figure 16A is a graph showing the CD4 / CD8 ratio at CART Cmax in peripheral blood versus the CD4 / CD8 ratio in DP CART+. [Figure 16B] This shows that LCAR-B38M is enriched in CD8 CAR+ T cells at the peak of proliferation in the bone marrow. Figure 16B is a graph showing the CD4 / CD8 ratio at CART Cmax in the bone marrow versus the CD4 / CD8 ratio in DP CART+. [Figure 16C] This shows that LCAR-B38M is enriched in CD8 CAR+ T cells at the peak of proliferation in the bone marrow. Figure 16C is a graph showing the correlation between the CD4 / CD8 ratio in peripheral blood and the CD4 / CD8 ratio in bone marrow on day 28. [Figure 17] The images show phenotypic changes in various T cell differentiation states: naive, stem memory (Tscm), central memory (Tcm), effector memory (Tem), effector (Teff), and effector memory RA (Temra). T cell activation and differentiation correlate with increased dependence on glycolysis and increased mitochondrial membrane potential, both of which may mediate the effector function of T cells in response to cancer. Images courtesy of Kiston, RJet al., Cell Metabolism, 2017, 26(1):94-109. [Figure 18]The phenotypic markers for each subset of LCAR-B38M CAR-T T cells are shown. Stem memory cells (Tscm) are CCR7+, CD45RO-, and CD95+. Central memory cells (Tcm) are CCR7+ and CD45RO+. [Figure 19] The results of phenotypic characterization of patient cells (in MMY2001) at pre-57 and -7 days before and post-infusion of MMY2001 CAR-T cells, as well as at 14, 21, 28, and 56 days after infusion, are presented. Both CAR-T+ and CAR-T- cell phenotypes were characterized at 14, 21, 28, and 56 days. It is clear that CAR-T+ cells were substantially more enriched in central memory cells (Tcm) compared to CAR-T- cells. At 21–28 and 56 days, CAR-T+ cells were more enriched in stem memory cells (Tscm) compared to Tcm cells. [Figure 20A] This shows the results of phenotypic characterization of CD8 T cells (CD27+) from various patients in the MMY2001 study at Cmax. In most patients, the majority of CAR+CD8+ T cells are central memory cells at Cmax. [Figure 20B] This shows the results of phenotypic characterization of CD4 T cells from various patients in the MMY2001 study at Cmax. Most CAR+CD4+ T cells are effector memory cells at Cmax. [Figure 20C] This graph shows that the percentage of CD8+CD450RO-CD27+ cells predicts the clinical response. Patients with CR or PRTD had a higher percentage of CD8+CD450RO-CD27+ cells than those with PR or NR. Source: Fraietta et al., 2018 Nature Medicine 24, 563-71. [Figure 20D] An example graph shows the correlation between the percentage of CD8 stem cell memory T cells (left panel) or naive T cells (right panel) in each patient grouped by clinical response. [Figure 21A]The graph shows the percentage of multiple myeloma cells versus total white blood cells over the study period. CD38+ MM cells and CD38dim MM cells are as shown in the figure. [Figure 21B] The graph shows the amount of MESF antigen detected in CD38+ and CD38dim BCMA and GPRC5D leukocytes over the study period, as well as the percentage of antigen-plus MM. CD38+ BCMA cells, CD38dim BCMA cells, CD38+ GPRC5D cells, and CD38dim GPRC5D cells are as shown in the figure. [Figure 21C] The graphs show the percentage of PD1+CAR+CD8+ T cells (as a percentage of CD8 CARs) and Treg cells (as a percentage of CD4 T cells), both representing individual patients over the study period. The results provide insights into CAR-T exhaustion and regulatory mechanisms. [Figure 22] Similar to bb2121, we provide data demonstrating that LCAR-B38M proliferation correlates with CRS grade (as measured by the Cmax of transgene copies per microgram of genomic DNA). [Figure 23] Two graphs are shown illustrating the levels of two serum pro-inflammatory cytokines, IL-6 and IFN-γ, in individual subjects over the study period. IL-6 levels increased in most patients after infusion. [Figure 24] Two graphs are shown illustrating the respective levels of the two serum pro-inflammatory cytokines, IL-10 and TNF-α, in individual subjects over the study period. Both levels increased in most patients after infusion. [Figure 25] Two graphs are shown illustrating the respective levels of the two serum pro-inflammatory cytokines, IL-2 and IL-2Rα, in individual subjects over the study period. [Figure 26A] This graph shows that IL-6 serum cytokine levels correlate with CRS. [Figure 26B] This graph shows that IL-6 serum cytokine levels do not correlate with clinical response. [Figure 27] The following is an example of data showing that IL-6 serum Cmax may correlate with peak LCAR-B38M. The left panel is a figure fitted from Fraietta et al., 2018 Nature Medicine 24, 563-71. [Figure 28] This example illustrates data showing that baseline sBCMA levels do not correlate with baseline bone marrow percentages of tumor cells. [Figure 29] We illustrate treatment protocols that include LCAR-B38M, demonstrating that LCAR-B38M is a "living drug" in the dynamic environment of each individual patient. [Figure 30] This exemplifies LCAR-B38M as a CAR-T cell therapy utilizing two BCMA-targeting domains. See D'Agostino Curr Hematol Malig Rep.2017;12:344, O'Connor J Exp Med.2004;199:91, Friedman Hum Gene Ther.2018;29:585, and Sanchez Br J Haematol 2012;158:727. BCMA = B cell maturation antigen, CD = cluster of differentiation, MM = multiple myeloma, NKG2D = natural killer group 2D, SLAMF7 = signaling lymphocyte activator family member 7, VHH = single variable domain on the heavy chain. [Figure 31] The following illustrates the overall response rates for LCAR-B38M treatment. a: PR or better, evaluated by an independent review committee; b: Best response, where the patient had no stable or progressive disease. CR = complete response, ORR = overall response rate, NGS = next-generation sequencing, PR = partial response, sCR = exact complete response, VGPR = very good partial response. [Figure 32] This illustrates how the LCAR-B38M drug product is enriched in effector memory T cells. Tcm = central memory T cell, Tem = effector memory T cell, Temra = terminally differentiated T cell, Tn = naive T cell. [Figure 33]Illustrate that LCAR-B38M exhibits variable proliferation and persistence. Detectable persistence in peripheral blood. gDNA = genomic DNA, LOQ = lower limit of quantification. [Figure 34] Illustrate a durable response after loss of persistence of LCAR-B38M in blood. [Figure 35] Illustrate that proliferation and persistence are not associated with the best response. Tlast = last study day when CAR-T cell levels exceed LOQ. [Figure 36] Illustrate the preferential proliferation of CAR+CD8 T cells in blood and bone marrow. DP = drug product before injection, WB = whole blood. [Figure 37] Illustrate that the CAR-T memory phenotype may be associated with clinical activity. 1Blaeschke Cancer Immunol Immunother 2018;67:1053. Tscm = stem central memory T cells. [Figure 38] Illustrate that LCAR-B38M shows preferential proliferation of CD8 central memory T cells. The analysis is at the peak of proliferation. [Figure 39] Illustrate that the response does not depend on the level of baseline BCMA expression. MESF = molecule of equivalent soluble fluorochrome. [Figure 40] Illustrate the conclusions of LCAR-B38M from study MMY2001. [Figure 41] Illustrate a durable response after loss of persistence of LCAR-B38M in blood. [Figure 42] Illustrate the CAR+CD4 / CD8 ratio at T cell Cmax (Tmax).

Mode for Carrying Out the Invention

[0040] A description of exemplary embodiments follows.

[0041] This disclosure also relates to related nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies, or These antigen-binding portions, as well as the pharmaceuticals related to immune cells and CAR-expressing T cells of the present invention. The composition is provided. A method for characterizing the administration regimen, dosage form, and CAR-T cell phenotype. Laws are also provided.

[0042] Some aspects of the present invention are described below with reference to examples provided for illustrative purposes only. To provide a complete understanding of the present invention, numerous specific details, relationships, and methods are described. Please understand that this is the case. However, those skilled in the art will know that the present invention is not a concrete detail of the present invention. It can be implemented without one or more of the following, or by other means, protocol It will be readily apparent that this can be carried out with reagents, cell lines, and animals. Several actions may occur in different order and / or in parallel with other actions or events. Therefore, the present invention is not limited by the order of the acts or events exemplified. Furthermore, the present invention In order to implement the method, all the exemplified actions, steps, or events are required. This is not the case. Many of the techniques and procedures described or referenced herein are the same as those of our company. It is well understood by those involved and is commonly used using conventional methods.

[0043] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein refer to the same technical terms, notations, and other scientific terms used herein. Words or technical terms have meanings that are ordinarily understood by those skilled in the art to whom this invention relates. This is intended. In some cases, terms that have a commonly understood meaning are used for clarification. and / or as defined herein for immediate reference, such definition herein Including it would necessarily represent a substantial difference from what is generally understood in the relevant technical field. No interpretation is appropriate. Terms such as those defined in commonly used dictionaries are not appropriate. , as having meanings consistent with those meanings in the context of the related technology, and / or It is further understood that this must be interpreted as otherwise defined herein. It will probably happen.

[0044] definition The terms "approximately" or "about" include the fact that the value falls within a statistically meaningful range. Such a range is within one decimal place of a given value or range, preferably within 50%, more preferably within 2. It may be within 0%, more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the words "approximately" or "about" is a specific system under study. It depends on the element and can be easily understood by those skilled in the art.

[0045] The term "antibody" refers to a monoclonal antibody (a full-length antibody with an immunoglobulin Fc region). Antibody compositions having polyepitope specificity (including chain antibodies or antibodies consisting only of full-length heavy chains), Deviant antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody cleavage. Contains fragments (e.g., Fab, F(ab')2, and Fv). "Immunoglobulin" (Ig) The term "antibody" is used interchangeably with "antibody" in this specification. This includes single-domain antibodies, such as antibodies consisting only of the heavy chain.

[0046] The terms "heavy chain-only antibody" or "HCAb" refer to antibodies that contain heavy chains but are not typically found in 4-chain antibodies. This refers to functional antibodies that lack the light chain necessary for proper functioning. (Camels, llamas, or alpacas) It is known that (k) produces HCAb.

[0047] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). An sdAb alone can bind to an antigen without pairing with the corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred to herein as "VHHs". Some VHHs may also be known as nanobodies. Camelid sdAbs are one of the smallest known antigen-binding antibody fragments (see, for example, Hamers-Casterman et al., Nature 363:446-8 (1993), Greenberg et al., Nature 374:168-73 (1995), Hassanzadeh-Ghas sabeh et al., Nanomedicine (Lond), 8:1013-2 6 (2013)). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site. Antibodies consisting only of heavy chains from camelid species are "VHHs".

[0048] ​​​​​​​​​It has a single heavy chain variable region called [name of region]. Therefore, VHH is a special type of VH. be.

[0049] The term "variable" refers to a situation where a specific segment of the variable domain has a significantly different sequence from antibody to antibody. This refers to the fact that the V domain mediates antigen binding, and the specific antibody against a particular antigen. Defining the opposite sex. However, its variability is not uniform across the entire span of the variability domain. It is not distributed. Instead, it is a hypervariable region in both the light chain and heavy chain variable domains. It is concentrated in three segments called hypervariable regions (HVRs). The more highly preserved portion of the domain is called the framework region (FR). The variable domains of the Eave heavy and light chains each contain four FR regions, and are mostly beta-sea It takes on a t-structure, connected by three HVRs, forming a loop that connects the beta-sheet structure. This is achieved, and in some cases forms part of the beta sheet structure. HVR in each chain is FR The regions are held together in close proximity, and using HVR from other chains, the antigen-binding site of the antibody Contributes to the formation of positions (Kabat et al., Sequences of Immu nological interest,Fifth Edition,Nationa l Institute of Health,Bethesda,Md.(1991) (See reference). The constant domain is not directly involved in the binding of the antibody to the antigen, but rather in antibody-dependent Antibodies are involved in various effector functions, such as cytotoxicity.

[0050] The terms "antibody fragment," "antibody fragment," "functional fragment of an antibody," and "antigen-binding portion" are also used. The term refers to one or more fragments or parts of an antibody that retain the ability to specifically bind to an antigen. As used interchangeably in this specification, (generally, Holliger et See al., Nat. Biotech., 23(9):1 126-1129 (2005) (See reference). The antigen recognition portion of the CAR encoded by the nucleic acid sequence of the present invention is optional. It may contain BCMA-conjugated antibody fragments. Preferably, the antibody fragments consist of, for example, one or two. The above CDR, variable region (or part thereof), steady region (or part thereof), or combination thereof This includes combinations. Examples of antibody fragments include (i) VL, VH, CL, and CHI domains. (ii) a monovalent fragment consisting of (ii) a Fab fragment, and a disulfide bridge in the hinge region. F(ab')2 fragment is a divalent fragment containing two linked Fab fragments, (iii) antibody Fv fragment consisting of VL and VH domains of a single arm of the body, (iv) two domains Linked by a synthetic linker, which allows it to be synthesized as a single polypeptide chain. A single-chain F is a monovalent molecule consisting of two domains of the Fv fragment (i.e., VL and VH). v (single chain Fv, scFv) (e.g., Bird et al.) Science, 242:423-426 (1988), Huston et al. Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988 ), and Osbourn et al., Nat. Biotechnol, 16:778 (See 1998), and (v) each polypeptide chain is the same polypeptide chain The peptide linker is too short to allow pairing between VH and VL above. It contains linked VH, thereby complementary doping on different VH-VL polypeptide chains. It promotes pairing between molecules, generating a dimer molecule with two functional antigen-binding sites. Examples include, but are not limited to, dimers of lipeptide chains called dibodies. The fragment is known in the art, for example, U.S. Patent Application Publication 2009 / 00930 This is described in detail in issue 24(A1).

[0051] The term "Cmax" in this specification refers to the time after the administration of the drug and the second dose. This is used to refer to the maximum concentration of effector CAR-T cells in the blood before administration. The reference to "at Cmax" in this context refers to the maximum number of effector CAR-T cells in the blood. This refers to the day on which the concentration is achieved in the target area.

[0052] As used herein, “specifically bind,” “specifically recognize,” or “to.” The term "specifically" refers to the presence of a heterogeneous population of molecules, including biological molecules. The bond between the target and the antigen-binding protein (CAR or sdAb, etc.) determines the presence of the target. This refers to measurable and reproducible interactions such as those between two or more people.

[0053] The term "specificity" refers to the antigen-binding protein (CAR or) of a specific epitope of an antigen. This refers to the selective recognition of antibodies (such as sdAb). Natural antibodies, for example, are monospecific. The term "heterosexual" refers to a pair of antigen-binding proteins (such as CARs or sdAbs) that are two or three in size. This indicates that the antigen-binding sites described above have at least two of them that bind to different antigens. As used herein, "bispecificity" refers to antigen-binding proteins (CAR or sd This indicates that (e.g., Ab) has two different antigen-binding specificities.

[0054] Chimeric antigen receptors (CARs) are antibodies linked to the T cell signaling domain (sc An artificially constructed hybrid protein or poly(Fv) containing the antigen-binding domain It is a lipeptide. A characteristic of CARs is that they target non-MHC restricted, selectively chosen targets. By redirecting T cell specificity and reactivity, and utilizing the antigen-binding properties of monoclonal antibodies... It can be said that this ability is achieved. Through non-MHC restriction antigen recognition, T cells expressing CARs Because it can recognize antigens independently of antigen processing, it is a major mechanism for tumor escape. It bypasses canism. Furthermore, when expressed in T cells, CAR is endogenous T cell receptor The alpha and beta chains of the T cell receptor (TCR) do not dimerize favorably. T cells expressing AR are, in this specification, CAR T cells, CAR-T cells, or CAR These are called modified T cells, and these terms are used interchangeably in this specification. The cells are, It can be genetically modified to stably express an antibody-binding domain on its surface. It confers novel antigen specificity that is MHC-independent. "BCMA CAR" is specific to BCMA. This refers to CARs that have a typical extracellular binding domain. "Double-epitope CARs" are BCMs. This refers to a CAR that has extracellular binding domains specific to two different epitopes of cell A.

[0055] "LCAR-B38M" is a combination of two B cell maturation inhibitors designed to confer binding activity. Chimeric antigen receptor T cells containing a proto(BCMA)-targeted single-domain antibody (chimeric a This is a CAR-T therapy (antigen receptor T cell therapy). LCAR-B38M is LCAR- May contain T lymphocytes transduced with B38M CAR, and CAR is a lentiviral vector Encoded by CAR. CAR is a target of human B cell maturation antigen (anti-BCMA CAR). The target is the lentiviral vector encoding LCAR-B38M CAR, as shown in the figure. Provided to 2A.

[0056] The terms "express" and "expression" refer to the generation of information about a gene or DNA sequence. To enable or generate, for example, the transcription and translation of a corresponding gene or DNA sequence. This means generating proteins by activating cellular functions involved in D NA sequences are expressed within or by cells to form "expression products" such as proteins. The expression product itself, for example, the resulting protein, is said to have been "expressed" by the cell. It is also possible to characterize the expression product as intracellular, extracellular, or transmembrane. ru.

[0057] The terms "to treat" or "treatment" do not mean delaying an unwanted physiological change or disease. To reduce, or to achieve beneficial or desired clinical outcomes during treatment. This refers to a therapeutic procedure whose purpose is to achieve a beneficial or desired clinical outcome, such as a detectable or Whether undetectable or not, it can alleviate symptoms, reduce the severity of the disease, and stabilize ( In other words, a disease state that does not worsen, a delay or slowing of disease progression, or improvement or alleviation of the disease state. , and remission (whether partial or complete) are also included. "Treatment" is Furthermore, the survival time was extended compared to the expected survival time if the subjects had not received treatment. This could mean causing unwanted physiological changes or Subjects who already have a disease, as well as subjects who have physiological changes or a tendency to develop a disease It is included.

[0058] As used herein, the term "subject" refers to an animal. The terms may be used interchangeably herein with respect to the subject. Thus, “subject” is , including, as patients, a person being treated for a disease or for the prevention of a disease. The method can be used to treat animal subjects belonging to any classification. Examples of such animals and Mammals are an example of this. Mammals include rodents such as mice and hamsters. This includes mammals, as well as lagomorphic mammals such as rabbits, but is not limited to these. Mammals may belong to the order Carnivora, which includes the families Felidae (cats) and Canidae (dogs). Mammals include Artiodactyla, which includes Bovidae (cattle) and Suidae (pigs), or Equidae (wild boars). It may be from the odd-toed ungulates, including (M). Mammals include the order Primates, Ceboids, Alternatively, they may belong to the Simoid order (monkeys) or the Haplorhini suborder (humans and great apes). In one embodiment, the mammal is a human.

[0059] The term “effective” when applied to a dose or amount means that it is administered to the target that requires it. This refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity. (Combination of active ingredients) When administering these drugs, the effective dose of the combination is the amount that would be effective if each drug were administered individually. Please note that the amount of the ingredient may or may not be present. The required amount depends on the species, age, and overall health of the subject, the severity of the condition being treated, and the specific method used. The appropriate course of action varies depending on the drug, the method of administration, and the individual patient.

[0060] The phrase "pharmaceutically acceptable" is used in reference to the compositions described herein. In some cases, it is physiologically tolerable and usually when administered to mammals (e.g., humans). This refers to molecular entities that do not cause adverse reactions and other components of such compositions. Preferably, the term "Medically acceptable" means that the use in mammals, more specifically in humans, is permitted by federal law. Approved by a government or state regulatory agency, or by the United States Pharmacopeia or other This means it is listed in a generally accepted pharmacopoeia.

[0061] The terms used herein are for the sole purpose of describing specific embodiments. This is not intended to be limiting. When used herein, unless otherwise specified in the context. Unless otherwise specified, the indefinite articles "a," "an," and "the" are understood to include multiple demonstrative pronouns. It should be done.

[0062] Throughout this disclosure, various aspects of this disclosure may be presented in scope form. The terms are provided solely for convenience and brevity and do not constitute a firm limitation on the scope of this disclosure. It should be understood that it should not be interpreted in any way. Therefore, the description of the scope is all possible. It should be considered that the possible partial range, as well as the individual numerical values ​​within that range, are specifically disclosed. For example, the explanation of ranges such as 1-6 is 1-3, 1-4, 1-5, 2-4, 2-6. , a specific disclosed sub-range such as 3 to 6, and individual numbers within that range, for example, 1 It should be considered to have 2, 2.7, 3, 4, 5, 5.3, and 6. Another example and Therefore, the range of 95-99% identity is 95%, 96%, 97%, 98%, or 99%. This includes items with % identity, such as 96-99%, 96-98%, 96-97%, and 97-9. This includes sub-ranges such as 9%, 97-98%, and 98-99% identity. It applies regardless of size.

[0063] vector The polynucleotide sequences encoding CARs described in this application were obtained using standard recombination techniques. It can be obtained using [this method]. The desired polynucleotide sequence is used in antimicrobial cells such as hybridoma cells. They can be isolated from somatic cells and sequenced. Alternatively, polynucleotides can be nucleotid. It can be synthesized using synthetic factors or PCR technology. Many promoters are known to be recognized in this way. Selected promoters are restricted The promoter was removed from the source DNA via enzymatic digestion, and the isolated promoter sequence was obtained. By inserting into the vector of this application, a cistron DN encoding a light chain or heavy chain can be created. It can be operably connected to A.

[0064] This disclosure also provides a vector comprising a nucleic acid sequence encoding the CAR of the present invention. The vectors are, for example, plasmids, cosmids, viral vectors (for example, retroviruses or The vector may be an adenovirus or a phage. Suitable vectors and methods for preparing vectors. This is well known in the relevant technical field (for example, Sambrook et al. 2001)Molecular Cloning: A Laboratory Manual al.3rd ed.Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, Ausube l et al. eds.(2005)Current Protocols in M olecular Biology.John Wiley and Sons,Inc (See .:Hoboken,NJ).

[0065] In addition to the nucleic acid sequence of the present invention that encodes CAR, the vector is preferably located inside a host cell. Promoter, enhancer, polyadenylation signal, providing expression of nucleic acid sequences. Transcription terminators, internal ribosome entry sites (IRs) Includes expression regulatory sequences such as ES. Exemplary expression regulatory sequences are known in the art. For example, Goddel, Gene Expression Technology gy:Methods in Enzymology,Vol.185,Academi This is described in c Press, San Diego, Calif. (1990).

[0066] Numerous promoters, including constitutive, inducible, and repressive promoters from various different sources. Motors are well known in the relevant technical field. As a typical source of promoters Examples include viruses, mammals, insects, plants, yeasts, and bacteria, and these A suitable promoter from the source is readily available, or, for example, ATCC. Based on which sediments, as well as the arrangements publicly available from other commercial or individual sources It can be synthesized. The promoter is unidirectional (i.e., opens transcription in one direction). (to start) or bidirectional (i.e., to start transcription in any of three or five directions) It is possible. Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD( araA) Bacterial expression system, cytomegalovirus (CMV) promoter Examples include the SV40 promoter and the RSV promoter. For example, the Tet system (U.S. Patent No. 5,464,758 and No. 5,811) (No. 4,618), Ecdysone-inducible system (No et al., Proc. Natl. Ac ad.Sci., 93:3346-3351 (1996)), T-REX (trademark) series (I nvitrogen, Carlsbad, CA), LACSWITCH(TM) series (St. ratagene (San Diego, CA), and Cre-ERT tamoxifen induce Conductive recombinase system (Indra et al., Nuc. Acid. Res., 27 :4324-4327(1999), Nuc.Acid.Res.,28:e99(20 00), U.S. Patent No. 7,112,715, and Kramer & Fussenegger Methods Mol. Biol, 308:123-144 (2005) is cited as an example. It can be done.

[0067] As used herein, the term "enhancer" means, for example, that it is activatable This refers to a DNA sequence that increases the transcription of nucleic acid sequences linked to it.

[0068] Enhancers can be located several kilobases away from the coding region of the nucleic acid sequence, and are regulatory factors. It can mediate the binding of offspring, the pattern of DNA methylation, or changes in DNA structure. Numerous enhancers from the supply source are well known in the art and have been cloned. As a polynucleotide, or a cloned polynucleotide (e.g., AT Available in deposits such as CC, as well as other commercial or individual sources. Several polynucleotides containing ter (such as the commonly used CMV promoter) Includes enhancer arrays. Enhancers are located upstream, internally, or downstream of the code array. It is possible. The term "Ig enhancer" refers to immunoglobulin (Ig). This refers to enhancer elements derived from enhancer regions mapped within a gene locus. Enhancers like these include, for example, heavy chain (mu) 5' enhancers and light chain (kappa) enhancers. ) 5' enhancer, kappa and mu-intron enhancer, and 3' enhancer - (Generally, Paul WE(ed), Fundamental I mmunology,3rd Edition,Raven Press,New Yo rk (1993), pages 353-363, and U.S. Patent No. 5,885,827 (See reference).

[0069] The vector may also contain a “selectable marker gene.” In other words, the term "selectable marker gene" refers to a nucleic acid sequence in the presence of a corresponding selector. nucleic acids that enable cells expressing them to be specifically selected for that purpose or against. Refers to a sequence. Suitable selectable marker genes are known in the art, for example, International Publication Nos. 1992 / 08796 and 1994 / 28143, Wigler e t al.,Proc.Natl.Acad.Sci.USA,77:3567(198 0), O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981), Mulligan & Berg, Proc. Natl. Ac. ad.Sci.USA,78:2072(1981), Colberre-Garapi n et al., J. Mol. Biol., 150:1 (1981), Santerr. e et al., Gene, 30:147 (1984), Kent et al., S science, 237:901-903(1987), Wigler et al.,C. ell, IP.223 (1977), Szybalska & Szybalski, Pro. c.Natl.Acad.Sci.USA,48:2026(1962), Lowy e t al., Cell, 22:817 (1980), and U.S. 5,122,46 It is described in items 4 and 5,770,359.

[0070] In some embodiments, the vector is an "episode" that can replicate within host cells. An "episome expression vector" or "episome" is used in the presence of appropriate selective pressure within a host cell. It persists as an extrachromosomal segment of DNA (e.g., Conese et al., See Gene Therapy, 11:1735–1742 (2004). A typical commercially available episome expression vector is Epstein-Var nuclear antigen 1 (EBN). A1) and epidemics using the origin of replication (oriP) of Epstein-Barr virus (EBV). Examples include, but are not limited to, somatic plasmids. Invitrogen(Ca Vectors pREP4, pCEP4, pREP7, and pcD from rlsbad,CA) NA3.1, and pB-CM from Stratagene (La Jolla, CA). V uses the T antigen and SV40 as the origin of replication instead of EBNAl and oriP. This represents a non-restrictive example of a somnal vector.

[0071] Other suitable vectors are integrated expression vectors that can be randomly integrated into the DNA of host cells. The vector is included, or it enables specific recombination between the expression vector and the host cell's chromosomes. It may contain a recombination site for the host cell's chromosomes. By utilizing endogenous expression regulatory sequences, it is possible to induce the expression of desired proteins. Site-specific An example of a vector to be integrated using this method is Invitrogen(Carlsba d,CA) flp-in derivatives (e.g., pcDNA(trademark)5 / FRT), or St pExchange-6 core vector from ratagene (La Jolla, CA) Examples of components of the cre-lox system that can be seen are host cell chromosomes. An example of a vector to be randomly integrated is, for example, Invitrogen(Carl pcDNA3.1 from sbad,CA (when introduced in the absence of the T antigen), and P pCI or pFNI OA(ACT)FL from romega (Madison, WI) EXI (trademark) is one example.

[0072] Viral vectors can also be used. A typical viral expression vector is adenow Virus-based vectors (e.g., Crucell, Inc., Leiden, The Net) Adenoviruses (Per.C6 family), lentiviruses (available from herlands) System vectors (e.g., Life Technologies (Carlsbad, CA)) lentiviral pLPl (from), and retroviral vectors (e.g., Strat pFB-ERV plus pCFB-EGSH from agene (La Jolla, CA) Examples include, but are not limited to, the viral vector. It is a lentiviral vector.

[0073] The vector comprising the nucleic acid of the present invention that encodes CAR can be used in any suitable prokaryotic or eukaryotic cell. It is introduced into host cells, including vesicles, that can express the CAR encoded by it. Obtain. Preferred host cells can grow easily and reliably, and have a reasonably fast growth rate. It has a degree of [presumably a specific characteristic] and an expression system that is well characterized, and can easily and efficiently transform or transmute. It can be affected.

[0074] As used herein, the term “host cell” means a cell that may contain an expression vector. It refers to a specific type of cell. The host cell is a eukaryotic cell, for example, a plant, animal, fungus, or It may be an algae, or a prokaryotic cell, such as a bacterium or protist. Host cell These may be cultured cells or primary cells, that is, directly isolated from an organism, such as a human. The host cells can be adherent or suspension cells, that is, cells that proliferate in a suspension. Suitable host cells are known in the art, for example, DH5a E.co. li cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HE29 Examples include 3 cells. For the purpose of amplifying or replicating recombinant expression vectors, host cells are used. This could be a prokaryotic cell, for example, a DH5a cell. Host cells are used for the purpose of producing recombinant CARs. The host cell may be a mammalian cell. The host cell is preferably a human cell. It can be any type of cell, originate from any type of tissue, and be at any developmental stage. It may be. In one embodiment, the host cell is a peripheral blood lymphocyte. phocyte, PBL), peripheral blood mononuclear cell (PBMC) , or it may be a natural killer (NK). Preferably, the host cell is These are natural killer (NK) cells. More preferably, the host cell is a T cell. Methods for selecting suitable mammalian host cells, as well as cell transformation, culture, amplification, and Methods for cleaning and purification are known in the art.

[0075] This disclosure describes an isolated nucleic acid expressing the nucleic acid sequence of the present invention encoding the CAR described herein. The present invention provides a host cell. In one embodiment, the host cell is a T cell. The T cell of the present invention is , cultured T cells, for example, primary T cells, or T cells from cultured T cell lines, or from mammals These could be any T cells, such as the T cells obtained. When obtained from mammals, T cells are This includes, but is not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or bodily fluids. (i) Can be obtained from numerous sources. T cells can also be concentrated or purified. Preferably, these are human T cells (for example, isolated from humans). T cells are CD4 + / CD8+ double-positive T cells, CD4+ helper T cells, e.g., Th, and Th2 cells , CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, This includes, but is not limited to, T cells and other cells at any developmental stage. In the application morphology, T cells are CD8+ T cells or CD4+ T cells. T cell lines are, for example, , American Type Culture Collection (ATCC, M anassas, VA) and German Collection of Micro Available from Organisms and Cell Cultures (DSMZ) Yes, for example, Jurkat cells (ATCC TIB-152), Sup-Tl cells (A TCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Examples include Karpas 45 cells (DSMZ ACC-545) and their derivatives. In another embodiment, the host cell is a natural killer (NK) cell. NK cells are NK cells are a type of cytotoxic lymphocyte that plays a role in the innate immune system. Defined as granular lymphocytes, they are a common lymphocyte system that further generates B lymphocytes and T lymphocytes. It constitutes a third type of cell differentiated from progenitor cells (e.g., Immunobiol ogy, 5th ed., Janeway et al., eds., Garland See Publishing, New York, NY (2001). NK details The cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. After maturation, N K cells circulate as large lymphocytes with characteristic cytotoxic granules. NK cells are It recognizes and kills certain abnormal cells (for example, certain tumor cells and virus-infected cells). This can be achieved and is considered important in innate immune defense against intracellular pathogens. As mentioned above regarding T cells, NK cells are any NK cells (cultured NK cells, for example) For example, primary NK cells, or NK cells from cultured NK cell lines, or NK cells obtained from mammals. It can be cells, etc. When obtained from mammals, NK cells can come from numerous sources (blood, (including, but not limited to, bone marrow, lymph nodes, thymus, or other tissues or bodily fluids) NK cells can be obtained from. NK cells can also be concentrated or purified. NK cells are preferably These are human NK cells (for example, those isolated from humans). NK cell lines include, for example, Amer ican Type Culture Collection (ATCC, Manass Available from as,VA), for example, NK-92 cells (ATCC CRL-240 7) Examples include NK92MI cells (ATCC CRL-2408) and their derivatives. It can be done.

[0076] The nucleic acid sequence of the present invention that encodes CAR is "transfected", "transformed", Alternatively, it may be introduced into cells by "transduction." When used herein, "trans" "Infection," "transformation," or "transduction" is performed using physical or chemical methods. This allows for the introduction of one or more exogenous polynucleotides into host cells. It refers to. Many transfection techniques are known in the relevant field, for example, Calcium phosphate DNA coprecipitation method (e.g., Murray EJ (ed.), Met hods in Molecular Biology,Vol.7,Gene Tra nsfer and Expression Protocols,Humana Pr See ess (1991); DEAE-dextran method; electroporation method Cationic liposome-mediated transfection method; Tungsten particle-enhanced microparticles Impact method (Johnston, Nature, 346:776-777 (1990)); and Strontium biphosphate DNA coprecipitation method (Brash et al., Mol. Cell) Biol., 7:2031-2034 (1987) is one example. Phage vectors Alternatively, the viral vector, after the growth of infectious particles in suitable packaging cells, enters the host cell. These can be implemented, and many of them are commercially available.

[0077] Chimeric antigen receptor International Patent Publication No. 2018 / 028647 is incorporated herein by reference in its entirety. U.S. Patent Publication No. 2018 / 0230225, in its entirety, is referred to herein by reference. It will be incorporated into it.

[0078] This disclosure describes a method for treating a target using cells that express chimeric antigen receptors (CARs). Provided. CARs are extracellular antibodies containing one or more single-domain antibodies (such as VHH). It contains an antigen-binding domain. In various embodiments, (a) cells containing anti-BCMA sdAb (b) an external antigen-binding domain, and (c) a transmembrane domain, and an intracellular signaling domain. CARs that target BCMA containing polypeptides (referred to as "BCMA C" in this specification) It provides (also called "AR"). In some embodiments, the anti-BCMA sdAb is These are camels, chimeras, humans, or humanized forms. In some embodiments, intracellular signaling The signaling domain is the major intracellular signaling domain of immune effector cells (such as T cells). It contains . In some embodiments, the major intracellular signaling domain is CD4. It originates from. In some embodiments, the intracellular signaling domain is used for co-stimulatory signal transmission. It includes a signaling domain. In some embodiments, the co-stimulatory signaling domain is CD27 , CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICO Ligands for S, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and It is derived from a co-stimulatory molecule selected from a group consisting of combinations thereof. In a particular embodiment, The transmembrane domain originates from CD137.

[0079] In some embodiments, BCMA CAR has a C-terminus of the extracellular antigen-binding domain and a membrane Hinge domains located between the transconducting domain and the N-terminus (e.g., CD8α hinge domain) The following are further included. In some embodiments, the BCMA CAR is located at the N-terminus of the polypeptide. It further includes a signal peptide (such as CD8α signal peptide). Several implementations Morphologically, the polypeptide has a CD8α signal peptide from the N-terminus to the C-terminus, and an extracellular anti- The protobinding domain, CD8α hinge domain, CD28 transmembrane domain, and CD28-derived The first co-stimulus signaling domain, and the second co-stimulus signaling domain derived from CD137. It includes domains and major intracellular signaling domains derived from CD4. In this embodiment, the polypeptide has a CD8α signal peptide from the N-terminus to the C-terminus, and cells External antigen-binding domain, CD8α hinge domain, CD8α transmembrane domain, CD137 The second co-stimulus signaling domain derived from, and the major intracellular sigma derived from CD3ζ It contains a single transmission domain. In some embodiments, the BCMA CAR has single specificity. Yes, in some embodiments, BCMA CAR is monovalent.

[0080] This application also applies to two or more (2, 3, A combination including, but not limited to, 4, 5, 6, or any one of these. A CAR having a portion is provided. In some embodiments, one or two of the joint portions One or more of these are antigen-binding fragments. In some embodiments, one or two of the binding portions are antigen-binding fragments. One or more of these include single-domain antibodies.

[0081] In some embodiments, the CAR (a) specifically binds to an antigen (such as a tumor antigen). Multiple (at least one of approximately 2, 3, 4, 5, 6, or more) (b) an extracellular antigen-binding domain including a binding portion, (c) a transmembrane domain, and (c) an intracellular signaling domain. Polyvalent (divalent, trivalent, or more valent) polypeptides containing a transmission domain It is a CAR (etc.).

[0082] In some embodiments, sdAb(multiple sdAbs, or a first sdAb and / or The binding portion, such as (including the second sdAb), is found in camels, chimeras, humans, or hominized organisms. Yes. In some embodiments, the binding portion or sdAb is a peptide bond or a peptide linkage. They are connected to each other via linkers. In some embodiments, each peptide linker is , approximately 50 or less (any one of approximately 35, 25, 20, 15, 10, or 5 or less, etc.) This is the length of the amino acid in the molecule.

[0083] In some embodiments, the CAR has a C-terminus of the extracellular antigen-binding domain and a transmembrane domain. It further includes a hinge domain (such as the CD8α hinge domain) located between the N-terminus and the nucleotide. In some embodiments, the CAR is a signal peptide located at the N-terminus of the polypeptide. It also contains (such as CD8α signal peptide).

[0084] While I don't want to be constrained by theory, polyvalent CAR, or the first anti-BCM A CAR containing an extracellular antigen-binding domain including an A-binding portion and a second BCMA-binding portion is Targeting multimeric antigens or antigens via synergistic binding by different antigen-binding sites It may be particularly suitable for enhancing binding affinity or binding activity. Improved binding activity The ratio is 4.0 × 10⁻¹⁰ per kilogram of the mass of the object. 5 ~1.0×10 6 Individual CAR-T Cells, or 3.0 × 10 7 ~1.0×10 8 Therapy, such as the range of doses of individual CAR-T cells. This allows for a substantial reduction in the dose of CAR-T cells required to achieve the desired effect. To obtain the equivalent effect, the unit price CAR such as bb2121 is 5 of these amounts. It may be necessary to administer the medication at ~10 times the normal dose. In various embodiments, the reduced dose range is CA Cytokine release syndrome (CRS) and other RT therapy It may offer a substantial reduction in potentially dangerous side effects.

[0085] Various binding sites in CAR as described herein (for example, the first anti-BCMA binding site and The extracellular antigen-binding domain (including the second BCMA-binding moiety) is accessed via a peptide linker. They can be connected to each other. In some embodiments, the connecting parts (such as sdAb) are any They are directly connected to each other without peptide linkers. Different binding sites (such as sdAb) The peptide linkers to be connected may be the same or different. The ins can also be connected to each other via peptide linkers.

[0086] The peptide linker in the CAR described herein may be of any suitable length. In several embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, Any of the following amino acid lengths: 30, 35, 40, 50, 75, 100, or more In some embodiments, the peptide linker is approximately 100, 75, 50, 40 ,35,30,25,20,19,18,17,16,15,14,13,12,11, The length of an amino acid is less than or equal to any of the following: 10, 9, 8, 7, 6, 5, or less. In some embodiments, the length of the peptide linker is about 1 amino acid to about 10 amino acids. Approximately 1 amino acid to approximately 20 amino acids, approximately 1 amino acid to approximately 30 amino acids, approximately 5 amino acids to approximately 1 5 amino acids, approximately 10 to approximately 25 amino acids, approximately 5 to approximately 30 amino acids, approximately 10 Amino acids ~ approximately 30 amino acids in length, approximately 30 amino acids ~ approximately 50 amino acids, approximately 50 amino acids ~ It consists of either approximately 100 amino acids, or between approximately 1 and 100 amino acids.

[0087] The CAR of this application may be directly or indirectly connected to an extracellular antigen-binding domain in a transmembrane structure. Includes the domain.

[0088] CAR may contain a T cell activation moiety. The T cell activation moiety may be derived from any suitable molecule. Or it may be any preferred portion obtained therefrom. In one embodiment, for example, T cell activity The activating portion includes a transmembrane domain. The transmembrane domain is any fraction known in the art. It may be any transmembrane domain derived from or obtained therefrom. For example, a transmembrane domain It may be obtained from or derived from the CD8α molecule or the CD28 molecule. CD8 is T It functions as a co-receptor for cell receptors (TCRs) and is mainly expressed on the surface of cytotoxic T cells. It is a transmembrane glycoprotein. The most common forms of CD8 are the CD8α and CD8P chains. It exists as a dimer composed of [components]. CD28 is expressed on T cells and is essential for T cell activation. It provides essential co-stimulatory signals. CD28 provides CD80 (B7.1) and CD86 (B7.1) 2) is the receptor. In preferred embodiments, CD8α and CD28 are human.

[0089] In addition to the transmembrane domain, the T cell activation region is located within the intracellular (i.e., cytoplasmic) T cell pathway. It further contains a Gnar signaling domain. The intercellular T cell signaling domain is the CD28 molecule. CD3 zeta (ζ) molecule or a modified version thereof, human Fc receptor gamma (FcRγ) ) chain, CD27 molecule, OX40 molecule, 4-1BB molecule, or other molecules known in the art. It may be obtained from or derived from intracellular signaling molecules. As discussed above... CD28 is an important T cell marker in T cell costimulation. Cd3ζ is a TCR. It associates with and produces a signal, and the immune receptor tyrosine system activation motif (immunoreceptor It contains tyrosine-based activation motifs (ITAM). Also known as CD137. 4-1BB transmits a strong co-stimulatory signal to T cells, promoting differentiation and the development of T lymphocytes. Enhances long-term survival period. In preferred embodiments, CD28, CD3ζ, 4-1BB, O X40 and CD27 are human.

[0090] The T cell activation domain of CAR encoded by the nucleic acid sequence of the present invention is any combination In combination, one of the aforementioned transmembrane domains and the aforementioned intercellular T cell domain It may contain any one or more of the signaling domains. For example, the nucleic acid of the present invention. The sequence includes the CD28 transmembrane domain, as well as the intracellular T cell signaling pathways for CD28 and CD3ζ. It may encode a CAR including a transmission domain. Alternatively, for example, the nucleic acid sequence of the present invention is C D8α transmembrane domain, as well as CD28, CD3ζ, and Fc receptor gamma (Fc receptor ga Includes mma, FcRy) chains and / or 4-1BB intracellular T cell signaling domains It can code CAR.

[0091] In some embodiments, a first BCMA binding portion and / or a second BCMA binding portion This is an anti-BCMA sdAb. In some embodiments, the first BCMA binding portion is The first anti-BCMA sdAb is, and the second BCMA binding site is the second anti-BCMA It is sdAb. In a particular embodiment, the first BCMA binding portion is QVKLEESGG GLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVA VIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPE DTAVYYCAARRIDAADFDSWGQGTQVTVSS (Sequence No. 1) It contains an acid sequence. In certain embodiments, the first BCMA binding moiety is CAGGTCAAA CTGGAAGAATCTGGCGGAGGCCTGGTGCAGGCAGGACGGA GCCTGCGCCTGAGCTGCGCAGCATCCGAGCACACCTTCAG CTCCCACGTGATGGGCTGGTTTCGGCAGGCCCCAGGCAAG GAGAGAGAGAGCGTGGCCGTGATCGGCTGGAGGGACATCT CCACATCTTACGCCGATTCCGTGAAGGGCCCGGTTCACCAT CAGCCGGGACAACGCCAAGAAGACACTGTATCTGCAGATG AACAGCCTGAAGCCGAGGACACCGCCGTGTACTATTGCG CAGCAAGGAGAATCGACGCAGCAGACTTTGATTCCTGGGGG Nucleic acid sequence CCAGGGCACCCAGGTGACAGTGTCTAGC (Sequence ID 2) Includes a polypeptide coded by.

[0092] In a particular embodiment, the second BCMA binding portion is EVQLVESGGGLVQAGG SLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAY YAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAAD Contains the amino acid sequence RKSVMSIRPDYWGQGTQVTVSS (SEQ ID NO: 3). In a particular embodiment, the second BCMA binding portion is GAGGTGCAGCTGGTGGA GAGCGGAGGCGGCCTGGTGCAGGCCGGAGGCTCTCTGAGG CTGAGCTGTGCAGCATCCGGAAGAACCTTCACAATGGGCT GGTTTAGGCAGGCACCAGGAAAGGAGAGGGAGTTCGTGGC AGCAATCAGCCTGTCCCCTACCCTGGCCTACTATGCCGAG AGCGTGAAGGGCAGGTTTACCATCTCCCGCGATAACGCCA AGAATACAGTGGTGCTGCAGATGAACTCCCTGAAACCTGA GGACACAGCCCTGTACTATTGTGCCGCCGATCGGAAGAGC GTGATGAGCATTAGACCAGACTATTGGGGGCAGGGAACAC The nucleic acid sequence AGGTGACCGTGAGCAGC (SEQ ID NO: 4) encodes the port Contains lipeptides.

[0093] In some embodiments, the first BCMA binding portion and the second BCMA binding portion are ped They are connected to each other via a peptide linker. In certain embodiments, a peptide linker It contains the amino acid sequence GGGGS (SEQ ID NO: 5).

[0094] In some embodiments, the CAR polypeptide is located at the N-terminus of the polypeptide. It further contains signal peptides. In some embodiments, the signal peptide is CD8α It originates from. In certain embodiments, the signal peptide is MALPVTALLLPLALL Includes the amino acid sequence of LHAARP (SEQ ID NO: 6). In certain embodiments, the signal peptide Chido is ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTG The nucleic acid sequence GCTCTGCTGCTGCACGCTGCTCGCCCT (Sequence ID 7) Therefore, it includes the encoded polypeptide.

[0095] In certain embodiments, the transmembrane domain is IYIWAPLAGTCGVLLLSLVI Contains the amino acid sequence of TLYC (SEQ ID NO: 8).

[0096] The transmembrane domain is ATCTACATCTGGGCGCCCTTGGCCGGGACT TGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACT A specific implementation including a polypeptide encoded by the nucleic acid sequence GC (sequence number 9) Status.

[0097] In some embodiments, the intracellular signaling domain is the primary domain of immune effector cells. It contains essential intracellular signaling domains. The intracellular signaling domains are derived from CD3ζ Several embodiments result. In some embodiments, the intracellular signaling domain is , comprising one or more co-stimulatory signaling domains. In certain embodiments, intracellular The signaling domain is RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA Contains the amino acid sequence of LPPR (SEQ ID NO: 10).

[0098] In certain embodiments, the intracellular signaling domain is AGAGTGAAGTTCAG CAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTG TACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC Contains polypeptide encoded by the nucleic acid sequence GCTAA (SEQ ID NO: 11). In this embodiment, the intracellular signaling domain is KRGRKKLLYIFKQPFMR Amino acid sequence of PVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12) Includes. In certain embodiments, the intracellular signaling domain is AAACGGGGCAG AAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(Sequence ID) It contains a polypeptide encoded by the nucleic acid sequence 13).

[0099] CAR polypeptides have a C-terminus of the extracellular antigen-binding domain and an N-terminus of the transmembrane domain. Several embodiments further include hinge domains located between them. In certain embodiments, The hinge domain is TTTPAPRPPTPAPTIASQPLSLRPEACRPAA Contains the amino acid sequence GGAVHTRGLDFACD (SEQ ID NO: 15). Specific Embodiments So, the hinge domain is ATGGCTCTGCCCGTCACCGCTCTGCTGC TGCCTCTGGCTCTGCTGCTGCACGCTGCTCGCCCT(code) It contains a polypeptide encoded by the nucleic acid sequence of 7).

[0100] In one embodiment, CAR is one or more of the elements listed in Table 1, It includes everything.

[0101] [Table 1]

[0102] Immunoeffector cell composition "Immune effector cells" are immune cells capable of performing immune effector functions. In some embodiments, immune effector cells are expressed at least in FcγRIII, Perform ADCC effector function. Examples of immune effector cells that mediate ADCC and These include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, and cytotoxicity. Examples include sexual T cells, neutrophils, and eosinophils. In some embodiments, immunoeffectors - The cell is a T cell. In some embodiments, the T cell is CD4+ / CD8-, C D4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or any combination thereof. In some embodiments, T cells express CAR and CD20+ or ​​CD19 +When it binds to target cells such as tumor cells, it produces IL-2, TFN, and / or TNF. In some embodiments, CD8+ T cells express CARs and bind to target cells. This lyses antigen-specific target cells.

[0103] Biological methods for introducing vectors into immune effector cells include DNA and RNA This includes the use of vectors. Viral vectors insert genes into mammalian cells, such as human cells. This has become the most widely used method for introducing the vector into immunoeffectors. Chemical methods for introducing into cells include polymer complexes, nanocapsules, microspheres, and Lipid bases including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples include colloidal dispersion systems such as serosol systems. A similar colloidal system is liposomes (e.g., artificial membrane vesicles).

[0104] In this specification, (a) a first BCMA that specifically binds to the first epitope of BCMA The binding site and the second BCMA binding site that specifically binds to the second epitope of BCMA (b) an extracellular antigen-binding domain, and (c) an intracellular signaling domain. Main, including polypeptides, including CAR, 3.0 x 10 7 ~1.0×10 8 individual A dosage form containing CAR-T cells is provided, and the first and second epitopes are different. In certain embodiments, the dosage form is 3.0 × 10 7 ~4.0×10 7 individual CAR-T cells Includes. In certain embodiments, the dosage form is 3.5 × 10 7 ~4.5×10 7 Individual CAR-T Contains cells. In certain embodiments, the dosage form is 4.0 × 10 7 ~5.0×10 7 individual CAR -Contains T cells. In certain embodiments, the dosage form is 4.5 × 10 7 ~5.5×10 7 Individual C Contains AR-T cells. In certain embodiments, the dosage form is 5.0 × 10⁶ 7 ~6.0×10 7 pieces Contains CAR-T cells. In certain embodiments, the dosage form is 5.5 × 10 7 ~6.5×10 7 Contains CAR-T cells. In certain embodiments, the dosage form is 6.0 × 10 7 ~7.0× 10 7 Contains CAR-T cells. In certain embodiments, the dosage form is 6.5 × 10 7 ~7. 5 x 10 7 Contains CAR-T cells. In certain embodiments, the dosage form is 7.0 × 10 7 ~ 8.0×10 7 Contains CAR-T cells. In certain embodiments, the dosage form is 7.5 × 10⁶ 7 ~8.5×10 7 Contains CAR-T cells. In certain embodiments, the dosage form is 8.0× 10 7 ~9.0×10 7 Contains CAR-T cells. In certain embodiments, the dosage form is 8. 5 x 10 7 ~9.5×10 7 Contains 1 CAR-T cell. In certain embodiments, the dosage form is 9.0×10 7 ~1.0×10 8 Contains 1 CAR-T cell. In some embodiments, The dosage form is 2 x 10 7 ~8×10 7 Contains 1 CAR-T cell. In some embodiments, The dosage form is approximately 1 x 10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 ,about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9×10 7 , or approximately 1 x 10 8 individual CAR -Contains T cells. In some embodiments, the dosage form is approximately 5.1 × 10⁻⁶ 7 , about 5.2×10 7 , about 5.25×10 7 , about 5.3×10 7 , about 5.4×10 7 , about 5.5×10 7 ,about 5.6 × 10 7 , about 5.7×10 7 , about 5.8×10 7 , or approximately 5.9 × 10 7 Individual CA Contains RT cells. In one embodiment, the dosage form is approximately 5.25 × 10 7 individual CAR-T cells include.

[0105] In some embodiments, (a) a first epitope of BCMA that specifically binds to the first epitope of BCMA. A second anti-BCMA bacterium that specifically binds to the second epitope of BCMA and an anti-BCMA bacterium that specifically binds to the second epitope of BCMA. (b) an extracellular antigen-binding domain containing CMA sdAb, and (c) a transmembrane domain. 3.0 × 10¹³ CAR containing a polypeptide containing an intracellular signaling domain. 6 ~ 1.0 × 10 8 A dosage form containing individual manipulated immune effector cells (such as T cells) is provided. Therefore, the first epitope and the second epitope are different. In certain embodiments, the dosage form is 3.0×10 7 ~4.0×10 7 Contains 1 CAR-T cell. In certain embodiments, dosage form 3.5 × 10 7 ~4.5×10 7 Contains 1 CAR-T cell. In certain embodiments, The dosage form is 4.0 × 10 7 ~5.0×10 7 Contains a number of CAR-T cells. In certain embodiments The dosage form is 4.5 × 10 7 ~5.5×10 7 Contains individual CAR-T cells. Specific implementation form In terms of dosage form, it is 5.0 × 10 7 ~6.0×10 7 Contains a specific number of CAR-T cells. In terms of application method, the dosage form is 5.5 × 10 7 ~6.5×10 7 Contains individual CAR-T cells. In this embodiment, the dosage form is 6.0 × 10 7 ~7.0×10 7 Contains 1 CAR-T cell. In a particular embodiment, the dosage form is 6.5 × 10 7 ~7.5×10 7 Contains In certain embodiments, the dosage form is 7.0 × 10 7 ~8.0×10 7 individual CAR-T cells Includes. In certain embodiments, the dosage form is 7.5 × 10 7 ~8.5×10 7 Individual CAR-T Contains cells. In certain embodiments, the dosage form is 8.0 × 10 7 ~9.0×10 7 individual CAR -Contains T cells. In certain embodiments, the dosage form is 8.5 × 10 7 ~9.5×10 7 Individual C Contains AR-T cells. In certain embodiments, the dosage form is 9.0 × 10 7 ~1.0×10 8 pieces It contains CAR-T cells. In some embodiments, the dosage form is 2 × 10 7 ~8×10 7 pieces It contains CAR-T cells. In some embodiments, the dosage form is approximately 1 × 10⁶ 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8× 10 7 , about 9×10 7 , or approximately 1 x 10 8 Contains several CAR-T cells. Several implementations In terms of dosage form, it is approximately 5.1 × 10 7 , about 5.2×10 7 , about 5.25×10 7 , about 5.3 ×10 7 , about 5.4×10 7 , about 5.5×10 7 , about 5.6×10 7 , about 5.7×10 7 , about 5.8×10 7 , or approximately 5.9 × 10 7 Contains 1 CAR-T cell. In one embodiment, The dosage form is approximately 5.25 x 10 7 Contains 1 CAR-T cell.

[0106] Pharmaceutical compositions and preparations This application allows for the invention of any one of the anti-BCMA single-domain antibodies, or the present invention. Modified immunoassay containing one of the listed CARs (such as BCMA CAR) A pharmaceutical composition comprising one of the generatrix cells and a pharmaceutically acceptable carrier Further provision is provided. The pharmaceutical composition is available in the form of a lyophilized preparation or an aqueous solution, with a desired degree of purity. Any of the immunoeffector cells described herein possessing any selection of pharmaceutically acceptable cells It can be prepared by mixing with a carrier, excipient, or stabilizer (Remingto n's Pharmaceutical Sciences 16th edition Osol, A. Ed. (1980).

[0107] The compositions described herein may be administered as part of a pharmaceutical composition comprising one or more carriers. The choice of carrier may be such that a specific nucleic acid sequence, vector, or CAR of the present invention is expressed. Host cells express the nucleic acid sequence, vector, or CAR of the present invention, as well as host cells expressing the nucleic acid sequence, vector, or CAR of the present invention. It is partially determined by the specific method used to administer it. Therefore, this There are various suitable formulations of the Ming dynasty's pharmaceutical composition. For example, the pharmaceutical composition contains a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate. Examples include thorium and benzalkonium chloride. Two or more may be selected at will. A mixture of preservatives may be used. The preservative or mixture thereof typically makes up about half of the total composition. It exists in amounts ranging from 0.0001% by weight to approximately 2% by weight.

[0108] Furthermore, a buffering agent may be used in this composition. Suitable buffering agents include, for example, citric acid. Examples include acids, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents may be used as an option. Buffering agent or so The mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[0109] A composition comprising the nucleic acid sequence of the present invention that encodes a CAR, or a host cell expressing a CAR, It can be formulated as an inclusion complex such as a cyclodextrin-containing complex, or as a liposome. Liposomes are host cells (e.g., T cells or NK cells) or nucleic acid sequences of the present invention. It may be useful for targeting specific tissues. Liposomes also have a half-life of nucleic acid sequences according to the present invention. It can be used to increase. Many methods exist, for example, Szoka et al., A nn. Rev. Biophys. Bioeng., 9:467 (1980), and the United States Patent No. 4,235,871, No. 4,501,728, No. 4,837,028, And for preparing liposomes such as those described in No. 5,019,369 It is available. This composition is used before the delivery of the composition of the present invention to the site to be treated, Sustained release, delayed release, and sustained release to ensure that it occurs with enough time to cause it. A delivery system can be used. Many types of discharge delivery systems are available, and those skilled in the art can utilize them. This is known. Such a system avoids repeated administration of the composition, thereby improving the subject and medical This can improve convenience for teachers, and for specific embodiments of the composition in the present invention This may be particularly preferable.

[0110] Treatment method This application further relates to methods and compositions for use in cell immunotherapy. In this embodiment, cell immunotherapy includes, but is not limited to, hematological malignancies and solid tumors. This method is for treating cancers that do not exist. This method is for adults, including subsets of all ages. It is suitable for the treatment of pediatric populations and can be used as any treatment option, including as a first-line or subsequent option. It can be used.

[0111] The anti-BCMA sdAb, CAR, and engineered immunoeffector compounds described herein. Any type of cell (such as CAR-T cells) can be used in methods to treat cancer.

[0112] In certain embodiments, CAR-T cells are approximately 4.0 × 10⁶ 5 ~5.0×10 5 individual cells / kg, 4.5×10 5 ~5.5×10 5 cells / kg, 5.0 × 10 5 ~6.0×1 0 5 individual cells / kg, 5.5 × 10⁻⁶ 5 ~6.5×10 5 cells / kg, 6.0 × 10 5 ~7.0×10 5 cells / kg, 6.5 × 10⁻⁶ 5 ~7.5×10 5 cells / kg, 7 .0 × 10 5 ~8.0×10 5 individual cells / kg, 7.5 × 10⁻⁶ 5 ~8.5×10 5 Individual details cells / kg, 8.0×10 5 ~9.0×10 5 cells / kg, 8.5 × 10 5 ~9.5× 10 5 cells / kg, 9.0 × 10 5 ~1.0×10 6 Administered at a dose of individual cells / kg In certain embodiments, CAR-T cells are approximately 7.1 × 10⁻⁶5 cells / kg, approximately 7 .2×10 5 Cells per kg, approximately 7.3 × 10⁻⁶ 5 Cells per kg, approximately 7.4 × 10⁻⁶ 5 individual cells / kg, approximately 7.5×10 6 cells / kg, approximately 7.6 × 10⁻⁶ 6 cells / kg, approximately 7 0.7×10 6 Cells per kg, approximately 7.8 × 10⁻⁶ 6 cells / kg, or approximately 7.9 × 10⁶ 6 It is administered at a dose of cells / kg. In one embodiment, CAR-T cells are approximately 7.5 × 1 0 6 They are administered at a dose of cells / kg. In certain embodiments, CAR-T cells are approximately 3 0~4.0×10 7 It is administered in doses of individual cells. In certain embodiments, CAR-T cells The cells are approximately 3.5-4.5 × 10 7 It is administered in doses of individual cells. In certain embodiments, C AR-T cells are approximately 4.0-5.0 × 10⁶ 7 It is administered in doses of individual cells. Specific implementations In this state, CAR-T cells are approximately 4.5-5.5 × 10⁻⁶ 7 It is administered in doses of individual cells. In one embodiment, CAR-T cells are approximately 5.0-6.0 × 10⁶ 7 Administer in doses of individual cells. In certain embodiments, CAR-T cells are approximately 5.5-6.5 × 10⁻⁶ 7 individual cells It is administered in a dose. In certain embodiments, CAR-T cells are approximately 6.0-7.0 × 10⁶ 7 It is administered in doses of individual cells. In certain embodiments, CAR-T cells are administered in doses of approximately 6.5-7. 5 x 10 7 They are administered in doses of individual cells. In certain embodiments, CAR-T cells are administered in doses of approximately 7 0~8.0×10 7It is administered in doses of individual cells. In certain embodiments, CAR-T cells The cells are approximately 7.5-8.5 × 10 7 It is administered in doses of individual cells. In certain embodiments, C AR-T cells are approximately 8.0-9.0 × 10⁶ 7 It is administered in doses of individual cells. Specific implementations In this state, CAR-T cells are approximately 8.5-9.5 × 10⁻⁶ 7 It is administered in doses of individual cells. In one embodiment, CAR-T cells are approximately 9.0-10.0 × 10⁶ 7 administered in doses of individual cells In some embodiments, CAR-T cells are given approximately 5.1 × 10⁶ 7 , about 5.2× 10 7 , about 5.25×10 7 , about 5.3×10 7 , about 5.4×10 7 , about 5.5×10 7 , about 5.6×10 7 , about 5.7×10 7 , about 5.8×10 7 , or approximately 5.9 × 10 7 individual They are administered in doses of cells. In one embodiment, CAR-T cells are approximately 5.25 × 10⁶ 7 individual It is administered according to the prescribed dose.

[0113] In certain embodiments, CAR-T cells are approximately 4.0 × 10⁶ in the subject. 5 ~5.0×1 0 5 cells / kg, 4.5 × 10⁻⁶ 5 ~5.5×10 5 cells / kg, 5.0 × 10 5 ~6.0×10 5 individual cells / kg, 5.5 × 10⁻⁶ 5 ~6.5×10 5 cells / kg, 6 .0 × 10 5 ~7.0×10 5 cells / kg, 6.5 × 10⁻⁶5 ~7.5×10 5 Individual details cells / kg, 7.0×10 5 ~8.0×10 5 individual cells / kg, 7.5 × 10⁻⁶ 5 ~8.5× 10 5 cells / kg, 8.0 × 10 5 ~9.0×10 5 cells / kg, 8.5 × 10 5 ~9.5×10 5 cells / kg, 9.0 × 10 5 ~1.0×10 6 individual cells / kg It is administered in a dose that yields the desired result. In certain embodiments, CAR-T cells are administered to a subject at approximately 7 .1×10 5 cells / kg, approximately 7.2 × 10⁻⁶ 5 Cells per kg, approximately 7.3 × 10⁻⁶ 5 individual cells / kg, approximately 7.4×10 5 Cells per kg, approximately 7.5 × 10⁻⁶ 6 cells / kg, approximately 7 0.6×10 6 Cells per kg, approximately 7.7 × 10⁻⁶ 6 Cells per kg, approximately 7.8 × 10⁻⁶ 6 individual Cells / kg, or approximately 7.9 × 10⁻⁶ 6 It is administered at a dose that yields individual cells / kg. Morphologically, CAR-T cells are approximately 7.5 × 10⁻⁶ in the subject. 6 yields individual cells / kg It is administered in doses of approximately 3.0–4 in the subject. In certain embodiments, CAR-T cells are administered in doses of approximately 3.0–4 in the subject. .0 × 10 7 It is administered in a dose that yields a certain number of cells. In certain embodiments, CAR-T cells The cells are approximately 3.5–4.5 × 10⁻⁶ in the subject. 7 It is administered in a dose that yields individual cells. In one embodiment, CAR-T cells number approximately 4.0-5.0 × 10⁶ in the subject. 7 individual cells They are administered in doses that produce the desired result. In certain embodiments, CAR-T cells are administered in a subject that yields approximately 4.5~5.5×10 7 It is administered in a dose that yields a certain number of cells. In certain embodiments, C AR-T cells were approximately 5.0–6.0 × 10⁶ in the subjects. 7 Administer in a dose that yields a single cell. In certain embodiments, CAR-T cells are found in a sample size of approximately 5.5–6.5 × 10⁶ cells in the subject. 7 It is administered in a dose that yields a certain number of cells. In certain embodiments, CAR-T cells are the target Approximately 6.0-7.0 × 10 7 It is administered in a dose that yields individual cells. Specific implementation In this state, CAR-T cells number approximately 6.5-7.5 × 10⁻⁶ in the subject. 7 Bringing forth individual cells It is administered in doses. In certain embodiments, CAR-T cells are administered in a dose of approximately 7.0–8 in the subject. .0 × 10 7 It is administered in a dose that yields a certain number of cells. In certain embodiments, CAR-T cells The cells are approximately 7.5–8.5 × 10⁻⁶ in the subject. 7 It is administered in a dose that yields individual cells. In a typical embodiment, CAR-T cells are present in the subject at approximately 8.0–9.0 × 10⁶ 7 individual cells They are administered in doses that produce the desired result. In certain embodiments, CAR-T cells are administered in a subject that yields approximately 8.5~9.5×10 7 It is administered in a dose that yields a certain number of cells. In certain embodiments, C AR-T cells are approximately 9.0-10.0 × 10⁶ in the target population. 7 In a dose that delivers individual cells In some embodiments, CAR-T cells are administered to a subject at a rate of approximately 5.1 × 10⁶ 7 , about 5.2×10 7 , about 5.25×10 7 , about 5.3×10 7, about 5.4×10 7 , about 5 0.5×10 7 , about 5.6×10 7 , about 5.7×10 7 , about 5.8×10 7 , or approximately 5.9 ×10 7 It is administered in a dose that yields a certain number of cells. In one embodiment, CAR-T cells are opposed In elephants, approximately 5.25 × 10 7 It is administered in a dose that yields a single cell.

[0114] The methods described herein treat a variety of cancers, including both solid and liquid tumors. It may be used for the treatment of multiple myeloma. In certain embodiments, this method may be used to treat multiple myeloma. The methods described herein are used in adjuvant settings or neoadjuvant settings. The first therapy, the second therapy, the third therapy, or chemotherapy, surgery, radiation, gene therapy, Immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, high As a combination therapy with other types of cancer therapies known in the relevant field, such as frequency ablation. It can be used.

[0115] In some embodiments, the cancer is multiple myeloma. In some embodiments, The disease is classified into stages I and II based on the Durie-Salmon staging system. Alternatively, it is stage III, and / or stage A or stage B multiple myeloma. In some embodiments, cancer is International Myeloma Wo Based on the International Disease Staging System published by the Engagement Group (IMWG) It is multiple myeloma of stage I, stage II, or stage III.

[0116] Host cells expressing the CAR coding nucleic acid sequence of the present invention, or the CAR coding nucleic acid sequence of the present invention Compositions containing vectors with rows can be administered orally, intravenously, intraperitoneally, subcutaneously, in the lungs, percutaneously, intramuscularly, or nasally. It is administered to mammals using standard administration techniques, including intracavitary, intrabuccal, sublingual, or suppository administration. Obtain. This composition is preferably suitable for parenteral administration. When used herein, The term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. Preferably, the composition is delivered peripherally to the whole body by intravenous, intraperitoneal, or subcutaneous injection. It is administered to mammals.

[0117] Host cells expressing the CAR coding nucleic acid sequence of the present invention, or the CAR coding nucleic acid sequence of the present invention A composition containing a vector with columns may be co-administered to mammals with one or more additional It can be administered together with a therapeutic agent. "Co-administration" means that one or more CARs of the present invention are administered together. In a state close enough to enhance the effect of the additional treatment, or vice versa. a combination comprising one or more additional therapeutic agents and host cells or vectors of the present invention. This means administering the product. In this regard, the host cells or vectors of the present invention. A composition containing the above may be administered first, and one or more additional therapeutic agents may be administered second. It could be either true or the other way around.

[0118] The CAR-expressing cells and at least one additional therapeutic agent described herein are simultaneously the same Alternatively, it may be administered in separate compositions or in succession. In the case of succession, this specification Administer the CAR-expressing cells described in the book first, then administer the additional drugs, or in the order of administration. The order can be reversed.

[0119] Host cells expressing the CAR coding nucleic acid sequence of the present invention, or the CAR coding nucleic acid sequence of the present invention When a composition containing a vector with columns is administered to a mammal (e.g., a human), the production of CARs occurs. The physical activity can be measured by any suitable method known in the art. According to the method, CAR binds to BCMA on multiple myeloma cells, and the multiple myeloma cells are destroyed. It is destroyed. The binding of CAR to BCMA on the surface of multiple myeloma cells is, for example, ELIS. Using any preferred method known in the art, including A and flow cytometry The ability of CARs to destroy multiple myeloma cells can be assayed, for example, by Kochen derfer et al., J. Immunotherapy, 32(7):689- 702 (2009), and Herman et al. J. Immunological Methods, 285(1):25-40(2004) describes cytotoxicity CAR can be measured using any suitable method known in the art, such as ssey. Biological activity also involves specific substances such as CD107a, IFN-γ, IL-2, and TNF. It can be measured by assaying itokine expression.

[0120] Characterization of T cell phenotype In some embodiments, the cell population of the CAR-T dosage form described herein is, for example, divided It contains T cells or populations of T cells at various stages of differentiation. As for the stages of T cell differentiation, the final stage of differentiation From the fewest to the most numerous, naive T cells, stem central memory T cells, and Terminal memory T cells, effector memory T cells, and terminal effector T cells For example, after antigen exposure, naive T cells proliferate, and memory T cells, such as stem cells, develop. They differentiate into primary memory T cells and central memory T cells, and then into effector memory cells. —Differentiates into T cells. When it receives appropriate T cell receptors, co-stimuli, and inflammatory signals, Memory T cells further differentiate into terminal effector T cells. For example, Restifo. Blood.124.4(2014):476-77, and Joshi et al.J See Immunol.180.3(2008):1309-15.

[0121] Naive T cells may have the following expression patterns of cell surface markers: CCR7+, C D62L+, CD45RO-, CD95-. Stem central memory T cells (Tscm) are The following cell surface markers may be expressed: CCR7+, CD62L+, CD4 5RO-, CD95+. Central memory T cells (Tcm) are the next cell surface markers. Possible expression patterns include: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (Tem) have the following expression patterns of cell surface markers Available: CCR7-, CD62L-, CD45RO+, CD95+. End effect pedal T thin Teff cells may have the following expression patterns of cell surface markers: CCR7-, CD 62L-, CD45RO-, CD95+. For example, Gattinoni et al. at.Med.17(2011):1290-7, and Flynn et al.Cli See n.Translat.Immunol.3(2014):e20. Figure 1 Figures 7 and 18 also show markers expressed in each of these and additional classes of T cells. .

[0122] While I don't want to be constrained by theory, the pre-injection T cell phenotype is similar to that of CAR+ T cells. Growth and persistence, toxicity profile, and clinical response may correlate. The product, DP, and the post-injection CD4:CD8 ratio and CAR-T memory phenotype are Furthermore, information regarding the proliferation and persistence of CAR+ T cells, their toxicity profile, and clinical response is also available. This may provide information. Immunophenotyping of CD4 and CD8 T cell subsets is possible for DP itself. Initial collection of patient T cells by apheresis, and various stages including various points in time after infusion. This can be done in stages (for example, to characterize subsets, activation states, and ratios).

[0123] In various embodiments, the CD8 of CD4+ CAR-T cells in CAR-T cell doses The ratio to +CAR-T cells is less than 4. In some embodiments, CAR-T cells The ratio of CD4+ CAR-T cells to CD8+ CAR-T cells at the dose of cells is 2. It is full. In some embodiments, the dose of CAR-T cells is CD4+CAR-T The ratio of cells to CD8+CAR-T cells is less than 1.8 in some embodiments. This relates to the difference between CD4+ CAR-T cells and CD8+ CAR-T cells in terms of CAR-T cell dose. The ratio is less than 1.7. In some embodiments, the dose of CAR-T cells The ratio of CD4+ CAR-T cells to CD8+ CAR-T cells is less than 1.6. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, In 15, 16, 17, 18, 19, or 20 patients, the dose of CAR-T cells was The median ratio of CD4+ CAR-T cells to CD8+ CAR-T cells is 1.6. It is complete. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 1 In 3, 14, 15, 16, 17, 18, 19, or 20 patients, CAR-T cells The median ratio of CD4+ CAR-T cells to CD8+ CAR-T cells at the dose is , approximately 1.54. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11 In 12, 13, 14, 15, 16, 17, 18, 19, or 20 patients, CA Ratio of CD4+ CAR-T cells to CD8+ CAR-T cells at RT cell dose The median is 1.54.

[0124] In various embodiments of the above method, this method involves CD4+CAR in the target at Cmax. - Assay the amount of T cells and increase the amount of CD8+ CAR T cells in the control group using Cmax. The steps further include CD4+ CAR-T cells at Cmax and CD4+ CAR-T cells at Cmax. The ratio to 8+CAR-T cells is less than 3.5. In some embodiments, Cma The ratio of CD4+ CAR-T cells to CD8+ CAR-T cells at Cmax in x is 2 It is less than 0.0. In some embodiments, the Cmax of CD4+ CAR-T cells is less than 0.0. The ratio to CD8+CAR-T cells in ax is less than 1.2. (Several embodiments) So, regarding CD4+ CAR-T cells at Cmax and CD8+ CAR-T cells at Cmax... The ratio is less than 0.8. In some embodiments, CD4+CAR- at Cmax The ratio of T cells to CD8+CAR-T cells at Cmax is less than 0.6. In that embodiment, CD4+CAR-T cells at Cmax and CD8+CAR-T cells at Cmax - The ratio to T cells is less than 0.4. In some embodiments, CD at Cmax The ratio of 4+CAR-T cells to CD8+CAR-T cells at Cmax is less than 0.3. Yes. In some embodiments, the Cmax of CD4+CAR-T cells is C The ratio to D8+CAR-T cells is approximately 0.3. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, Or in 20 patients, the C of CD4+ CAR-T cells at CAR-T cell doses. The median ratio to D8+CAR-T cells is less than 1.6. In certain embodiments, At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 In 8, 19, or 20 patients, CD4+ CAR-T cells at Cmax showed CD8+ The median ratio to CAR-T cells is approximately 0.35. In certain embodiments, less Tomo 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 In 9 or 20 patients, CD4+ CAR-T cells at Cmax showed CD8+ CAR - The median ratio to T cells is 0.35. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or In 20 patients, CD4+ CAR-T cells at Cmax were compared with CD8+ CAR-T cells. The median ratio to is approximately 0.3. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 people In patients, the relationship between CD4+ CAR-T cells and CD8+ CAR-T cells at Cmax The median of the ratio is 0.3.

[0125] In various embodiments of the above method, central memory CAR+ T cells are administered in doses. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 ,34,35,36,37,38,39,40,41,42,43,44,45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6 At least 85% of the total CAR+ T cell volume after 0, 61, 62, 63, 64, or 65 days It constitutes. In some embodiments, central memory CAR+ T cells are administered The given numbers 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3 3, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 ,47,48,49,50,51,52,53,54,55,56,57,58,59, At least 90% of the total amount of CAR+ T cells after 60, 61, 62, 63, 64, or 65 days. It constitutes %. In some embodiments, central memory CAR+ T cells are dosed 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 were administered. ,20,21,22,23,24,25,26,27,28,29,30,31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 After a few days, they constitute at least 95% of the total amount of CAR+ T cells. In some embodiments, Central memory CAR+ T cells were administered at doses 7, 8, 9, 10, 11, and 12. , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, At least 97% of the total CAR+ T cell mass is formed after 26, 27, 28, 29, or 30 days. To accomplish.

[0126] In a particular embodiment, central memory CAR+ T cells are the total amount of CAR+ T cells 85-86%, 86-87%, 87-88%, 88-89%, 89-90%, 90-91% %, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%, 96- It consists of 97%, 97-98%, and 98-99%.

[0127] In various embodiments of the above method, effector memory CAR+ T cells are administered in doses. The given numbers 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3 3, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 ,47,48,49,50,51,52,53,54,55,56,57,58,59, At least 2% of the total amount of CAR+ T cells after 60, 61, 62, 63, 64, or 65 days It constitutes. In some embodiments, effector memory CAR+ T cells are administered in doses 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 were administered. ,20,21,22,23,24,25,26,27,28,29,30,31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 At least 5% of the total amount of CAR+ T cells after 60, 61, 62, 63, 64, or 65 days. It constitutes %. In some embodiments, effector memory CAR+ T cells are dose The drugs were administered to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 1. 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 4 After 5 days, they constitute at least 7% of the total amount of CAR+ T cells. In some embodiments, Effector memory CAR+ T cells were administered at doses 7, 8, 9, 10, 11, and 1. 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 At least 8% of the total amount of CAR+ T cells is formed on days 26, 27, 28, 29, or 30. To accomplish.

[0128] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. In the central memory, the total amount of CAR+CD8+T cells is compared to the total amount of CAR+CD8+T cells. The procedure further includes the step of assaying the ratio, where Central Memory CAR+ CD8+ T cells constitute at least 30% of the total number of CAR+CD8+ T cells. In several embodiments, central memory CAR+CD8+ T cells are CAR+CD8+ It constitutes at least 50% of the total T cell population. In some embodiments, the central memo Lee CAR+CD8+ T cells make up at least 50% of the total number of CAR+CD8+ T cells. In some embodiments, central memory CAR+CD8+ T cells are CA Constituting at least 80% of the total R+CD8+ T cells. In certain embodiments, Memory CAR+CD8+ T cells account for 55-70% of the total number of CAR+CD8+ T cells. 56-71%, 57-72%, 58-73%, 59-74%, 60-75%, 61-7 6%, 62-77%, 63-78%, 64-79%, 65-80%, 66-81%, 67 ~82%, 68~83%, 70~85%, 72~87%, 74~89%, 76~91%, It makes up 78-93% or 80-100%.

[0129] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. In relation to the total amount of CAR+CD4+T cells in central memory, The procedure further includes the step of assaying the ratio, where Central Memory CAR+ CD4+ T cells constitute at least 60% of the total number of CAR+CD4+ T cells. In several embodiments, central memory CAR+CD4+ T cells are CAR+CD4+ It constitutes at least 65% of the total T cell population. In some embodiments, the central memo Lee CAR+CD4+ T cells make up at least 70% of the total number of CAR+CD4+ T cells. In some embodiments, central memory CAR+CD4+ T cells are CA It constitutes at least 75% of the total amount of R+CD4+ T cells. In certain embodiments, Memory CAR+CD4+ T cells account for 65-80% of the total CAR+CD8+ T cell population. 66-81%, 67-82%, 68-83%, 69-84%, 70-85%, 71-8 6%, 72-87%, 73-88%, 74-89%, 75-90%, 76-91%, 77 ~92%, 78~93%, 80~90%, 82~92%, 84~94%, 86~96%, It makes up 88-98% or 90-100%.

[0130] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. The total amount of CAR+CD8+T cells in the effector memory The process further includes an assay step regarding the ratio, where the effector memory CA R+CD8+ T cells constitute at least 2% of the total number of CAR+CD8+ T cells. In some embodiments, effector memory CAR+CD8+T cells are CAR+CD It constitutes at least 5% of the total number of 8+ T cells. In some embodiments, effectors Memory CAR+CD8+ T cells account for at least 8% of the total number of CAR+CD8+ T cells. To constitute. In some embodiments, effector memory CAR+CD8+ T cells, They constitute at least 10% of the total number of CAR+CD8+ T cells.

[0131] In various embodiments of the above method, the method involves administering a dose and then targeting the subject at Cmax. The total amount of CAR+CD4+T cells in the effector memory The process further includes an assay step regarding the ratio, where the effector memory CA R+CD4+ T cells constitute at least 70% of the total number of CAR+CD4+ T cells. In some embodiments, effector memory CAR+CD4+ T cells are CAR+C It constitutes at least 75% of the total number of D4+ T cells. In some embodiments, the effect Ter memory CAR+CD4+ T cells are at least 8 of the total number of CAR+CD4+ T cells. It constitutes 0%. In some embodiments, the effector memory CAR+CD4+T is fine Cells constitute at least 90% of the total number of CAR+CD4+ T cells. In certain embodiments, The effector memory CAR+CD4+T cells are the total amount of CAR+CD8+T cells. 70-80%, 70-85%, 71-86%, 72-87%, 73-88%, 74-89 %, 75-90%, 76-91%, 77-92%, 78-93%, 80-90%, 82- It consists of 92%, 84-94%, 86-96%, 88-98%, or 90-100%.

[0132] While I don't want to be constrained by theory, the percentage of cells in patients is, As shown in Figures 22C and 22D, clinical responses can be predicted.

[0133] Cytokines (e.g., IL-6, IFN-γ, IL-10, TNF-α, IL-2, The assay for IL-2Rα is performed at various points in time during treatment, for example, when administering drug products (DP). Ten days before administration or infusion, at the time of administration or infusion of DP, and at any time after administration of DP (for example) (Then, the procedure is performed on 10, 20, 30, 40, 60, 80, and 100 days after DP administration.) It is possible. I do not wish to be bound by theory, but IL-6 serum cytokine level As shown in the data in Figures 26A and 26B, the correlation with cytokine release syndrome is This is possible, but it does not correlate with clinical response.

[0134] Kits and manufactured articles Any of the compositions described herein may be included in the kit, and in some embodiments The kit provides engineered immortalized CAR-T cells, which can also be cultured in a culture medium. It may contain reagents suitable for propagation.

[0135] In non-specific examples, chimeric receptor expression constructs, chimeric receptor expression constructs One or more reagents for generating the expression construct, transfection of the expression construct Cells for and / or transfection of expression constructs for immortality One or more instruments for obtaining T cells (such instruments include syringes, pipettes, pins) This may be a set and / or any such medically approved device.

[0136] In some embodiments, the kit includes reagents or apparatus for electroporation of cells. Includes.

[0137] In some embodiments, the kit includes artificial antigen-presenting cells.

[0138] The kit contains one or more preferably equally divided compositions of the present invention or compositions of the present invention. The kit may include reagents for production. The components of the kit are in aqueous medium or lyophilized form. It may be packaged in either way. The kit container means at least one vial This may include bottles, test tubes, flasks, syringes, or other container means, and within them Components may be added, and preferably, they may be preferably divided into equal parts. Two or more in the kit. If the component is present, the kit can also generally include the additional component separately. This may include a second, third, or other additional container. However, the composition of the components may vary. Various combinations may be included in the vial. The kit of the present invention is typically a chimera. Means for housing the receptor construct and any other sealed packaging for commercial sale This will include reagent containers. Such containers may, for example, hold the desired vials. Examples include injection-molded or blow-molded plastic containers. [Examples]

[0139] To further illustrate some of the embodiments disclosed herein, the following examples are provided. The examples provided are not intended to limit the disclosed embodiments, but rather to illustrate them. ru.

[0140] (Example 1) LCAR-B38M (JNJ-4528) is designed to confer binding activity. Chimeric antigen receptor T1 containing a single-domain antibody targeting B cell maturation antigen (BCMA) This is a cell-mediated (CAR-T) therapy. Figure 2 shows a map of the construct and a schematic diagram of LCAR-B38M. As shown in A. L used to produce the LCAR-B38M cells tested herein. The CAR-B38M construct includes the sequences listed in Table 2.

[0141] [Table 2]

[0142] Apheresis samples were collected from 25 patients, T cells were selected, and LCAR-B38M was used. Transduced with a lentivirus encoding a BCMA CAR construct that expresses CAR. A schematic diagram of the experiment is shown in Figure 2B. Next, as outlined in Figure 3, 25 patients underwent M This study followed the MY2001 study. The patient population consisted of patients with relapsed multiple myeloma, or PI / IMiD and Three previous treatment options or dual refractory treatment for previous PI, IMiD, and αCD38 exposure This included patients with refractory multiple myeloma. The primary objective of the MMY2001 study was to include those with refractory multiple myeloma. This involved the safety and establishment of RP2D, including studies on the incidence and severity of adverse events (Phase 1). Phase 1b). Another primary objective is effectiveness: partial response as defined by the IMWG. Objective Response Rate (PRA) ORR) (Phase 2). The second objective is: incidence and severity of adverse events (Phase 2). ), immunogenicity assessment, post-treatment PRO and HRQoL assessment, PK and PD characterization, and Further evaluation of efficacy characteristics as needed. Clinical responses from patients are summarized in Figure 4.

[0143] T cells were transduced and proliferated for a period of approximately 3-4 weeks, but in 25 patients... , bridging therapy (if necessary), and pre-treatment with cyclophosphamide and fludarabine. A treatment regimen was performed. During this period, various assays were performed on patient T cells. Post-proliferation Both apheresis samples and transduction cells are immune cell compositions (referred to as "drug products" in this specification). The (referred to as "DP") is evaluated by multiparametric flow cytometry. did.

[0144] The median percentage of CAR+ T cells in DP is 16% of the total cells (ranging from 6% to 28%). The median percentages are 12% (in the range of 4-22%) for CD4+CAR+ and CD8+C In AR+ T cells, the percentage was 7% (ranging from 3% to 20%). CD4 in CAR+ T cells during DP The :CD8 ratio was assayed, and the results are shown on the X-axis in Figure 14B. As shown in Figure 19, patients The phenotype of T cells was also assayed before infusion. Significant inter-patient variability was observed in T cell subsequences. (i.e., Naive, Tscm, Tcm, Tem, Teff, and Temra) This was observed in the composition of DP related to each patient, but this profile is different from CAR- and The results were similar in the CAR+ T cell subpopulation.

[0145] 0.75 × 106 Target dose of CAR+ T cells / kg (0.5~1.0 × 10⁻¹⁰) 6 A single infusion of LCAR-B38M DP was administered within the target range. (Table 3 (Phase 1b)) (See reference). Among 21 patients with post-baseline disease assessment, the overall response rate was The median follow-up period was 3 months (range 1-10), which was 91%. evaluable at 28 days post-injection by next-generation flow cytometry and / or next-generation sequencing. Among 15 patients with bone marrow (BM) samples, 10 out of 10 -5 sensitivity Level, 2 people are 10 -4 The three individuals had minimal residual disease, with a negative result at the sensitivity level, and the condition remained unconfirmed. They had clones. All patients were evaluated by flow cytometry. BCMA expression in BM tumor cells was observed at baseline, but levels varied among patients. Clinical response appears to be independent of BM BCMA expression.

[0146] Cmax, and the degree of proliferation and persistence of LCAR-B38M in whole blood over the study period. The degree was assayed. Although we do not wish to be bound by theory, Cmax is the response (hold It is related to the duration or depth of cell proliferation. The degree of sufficiency and deficiency of cell proliferation at the peak is related to dose escalation. It can provide information for improvement. Durability is related to the response (duration or depth). The degree of sufficiency and deficiency of the drug may provide information for improving the dosage regimen. The characteristics of the product (DP) can also provide information regarding its growth or persistence.

[0147] As shown in Figure 8B, CAR+ T cells proliferate after injection, and blood samples taken 10-14 days after injection are available. Twenty to eighty-seven percent of all T cells in the reached their peak. As shown in Figure 8A, microgram g A qPCR assay showing the number of transgene copies per DNA was also performed. Cmax is variable among patients but Tmax is consistent among patients. The CD4:C D8 ratio and the proportion of T cell memory subsets during final DP did not correlate with the proliferation of peak CAR+ T cells The proliferation of peak CAR+ T cells did not correlate with the response. As shown in Figures 8A to 8C In 15 / 28 patients who had at least 11 weeks of follow-up, the number of CA R+ T cells / μl was <LOQ (2 cells / μl) at week 11. Although preliminary there was no difference in the response rate between these patients compared to those with measurable CAR+ T cells after 8 weeks As shown by the data in Figures 10A, 10B, and 11B A similar trend was observed when proliferation and persistence were evaluated by measuring the transgene level Unlike the comparator bb2121CAR, there is no correlation between clinical response and either C max or persistence.

[0148] Both CD4+ and CD8+ CAR+ T cells proliferated in vivo, but the CD4:CD8 CAR+ ratio decreased at peak proliferation compared to final DP (from a median of 1 .54 to 0.35 as shown in Figure 14B), indicating preferential proliferation of CD8+ CAR+ T cells in the blood Calculations based on exploratory analysis showed that the CD4 / CD8 ratio at the time of CAR+ T cell Cmax (Tmax) had a median of 0.29 and a range of 0.08 to 3.4 (see Figure 42). At peak proliferation, CD8+ CAR+ T cells were mainly centra See Figure 42). At peak proliferation, CD8+ CAR+ T cells were mainly centra Memory phenotype (Tcm) (CCR7+CD45RO+, as shown in Figure 20A, 90 The median percentage [ranging from 29.3% to 98.5%] was shown. In contrast, the CD4+CAR+T detailed data... Cells, in peak proliferation, are effector memory (Tem) cells (CCR7-CD45 RO+, as shown in Figure 20B, is concentrated within a median of 87% [range of 69.5-98.1%]. It was shrinking. The CD4:CD8 ratio and the T cell memory subset composition were similar. The trend was observed in BM in all 11 patients who had evaluable samples on day 28.

[0149] As shown in Figures 20A and 20B, CD8+ CAR-T cells have stem memory (Tsc m): The Tcm subset showed a ratio of approximately 50:50, but CD4+CAR-T cells were T The cm:Tem subset shows a ratio of approximately 50:50, which corresponds to CD4+ and CD8+CA The differential T cell maturation process for R+ and CAR-T cells was shown.

[0150] The ratio of CD8+CD450RO-CD27+ cells is as shown in Figure 20C, clinical Predict the response. Patients with CR or PRTD have a higher response rate than those with PR or NR. It had a certain percentage of CD8+CD450RO-CD27+ cells. Figure 20D shows the clinical CD8 stem cell memory T cells in each patient, grouped by bed response (left panel) An example graph showing the correlation between the percentages of ) or naive T cells (right panel) is provided. .

[0151] The ratio of multiple myeloma cells to total leukocytes was assayed throughout the study period, and the results This is shown in Figure 21A. By day 56, the ratio of multiple myeloma cells to total white blood cells was, The ratio fell to a level below that of the first hour (day 0).

[0152] Peripheral pro-inflammatory cytokine levels are related to CAR+ T cell proliferation, T cell subsets, and It was assumed that these correlated with the patient toxicity profile. CRS and HLH / MAS were at high levels. It may correlate with peripheral cytokines (IL-6, IL2-RA). IL-6 levels are related to CA It may correlate with the peak proliferation of RT cells (Fraietta et al., 2018 N Ature Medicine 24, 563-571). Selected serum cytokines. This may indicate MoA and T cell subset frequencies. CAR-T cell proliferation and CRS initiation We tested the evaluation of peripheral cytokines in CAR+T cells. As shown in Figures 23-25, Cell proliferation peaks around day 10, and serum cytokine levels (i.e., IL-6) increase. This correlates with an increase in IFN-γ and IL-10, which coincides with the maximal proliferation of CAR+ T cells. As shown in the assay in Figure 22, proliferation of LCAR-B38M and cytokine release syndrome occurred. A correlation was observed between the symptom grade and the symptoms. In general, several pro-inflammatory cytokines An increase in IL-6 (i.e., IL-6) is associated with cytokine release syndrome, as shown in Figure 26A. It correlated with the onset of symptoms (median onset time over 7 days [range: 2 to 12 days]).

[0153] An additional assay was performed: PD1+CAR+CD8 compared to CD8CAR. Assay of the percentage of +T cells, and assay of the percentage of CD4 T cells. This was performed for several days throughout the entire study period for each patient, and the results are shown in Figure 21C. This could provide insights into CAR-T fatigue and regulatory mechanisms.

[0154] The above findings suggest that LCAR-B38M has a relatively low risk compared to other CAR-T therapies. This suggests that it is a differentiated CAR-T cell therapy that is highly active in large quantities. While we do not wish this to happen, the high activity of LCAR-B38M at relatively low doses suggests that... Preferential and consistent in vivo amplification of CD8+CAR+ T cells exhibiting central memory phenotype Potentially related to reproduction.

[0155] Table 3 below shows the results of the MMY2001 study and related Phase 2 studies presented above. This section summarizes the injection procedure performed in the study.

[0156] [Table 3]

[0157] All patents, published patents, and teachings cited herein are to be taken in their entirety. It is incorporated by illumination.

[0158] Exemplary embodiments have been specifically shown and described, but those skilled in the art will see the attached claims. Without departing from the scope of embodiments included in the scope, various forms and details Understand that changes may be made.

[0159] array LCAR-B38M CD8α signal peptide, CD8α SP amino acid MALPVTALLLPLALLLHAARP(Sequence ID 6) LCAR-B38M BCMA-binding domain, VHH1 amino acid sequence QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQ APGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTL YLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSS (Sequence ID 1) LCAR-B38M BCMA-binding domain, G4S linker amino acid sequence GGGGS (Sequence No. 5) LCAR-B38M BCMA-binding domain, VHH2 amino acid sequence EVQLVESGGGLVQAGGSLLRLSCAASGRTFTMGWFRQAPG KEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQ MNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSS( Sequence ID 3) LCAR-B38M CD8α Hinge Amino Acid Sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACD (Sequence ID 15) LCAR-B38M CD8α transmembrane amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (Sequence ID 8) LCAR-B38M CD137 cytoplasmic amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CEL (Sequence ID 12) LCAR-B38M CD3ζ cytoplasmic amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 10 ) LCAR-B38M CD8α signal peptide CD8α SP nucleic acid sequence ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCT CTGCTGCTGCACGCTGCTCGCCCT(Sequence ID 7) LCAR-B38M BCMA-binding domain, VHH1 nucleic acid sequence CAGGTCAAACTGGAAGAATCTGGCGGAGGCCTGGTGCAG GCAGGACGGAGCCTGCGCCTGAGCTGCGCAGCATCCGAGC ACACCTTCAGCTCCCACGTGATGGGCTGGTTTCGGCAGGC CCCAGGCAAGGAGAGAGAGAGCGTGGCCGTGATCGGCTGG AGGGACATCTCCACATCTTACGCCGATTCCGTGAAGGGCC GGTTCACCATCAGCCGGGACAACGCCAAGAAGACACTGTA TCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTG TACTATTGCGCAGCAAGGAGAATCGACGCAGCAGACTTTG ATTCCTGGGGCCAGGGCACCCAGGTGACAGTGTCTAGC(arrangement Column number 2) LCAR-B38M BCMA-binding domain, G4S linker (SEQ ID NO: 5) nucleic acid sequence GGAGGAGGAGGATCT (Sequence No. 16) LCAR-B38M BCMA-binding domain, VHH2 nucleic acid sequence GAGGTGCAGCTGGTGGAGAGCGGAGGCGGCCTGGTGCAG GCCGGAGGCTCTCTGAGGCTGAGCTGTGCAGCATCCGGAA GAACCTTCACAATGGGCTGGTTTAGGCAGGCACCAGGAAA GGAGAGGGAGTTCGTGGCAGCAATCAGCCTGTCCCCTACC CTGGCCTACTATGCCGAGAGCGTGAAGGGCAGGTTTACCA TCTCCCGCGATAACGCCAAGAATACAGTGGTGCTGCAGAT GAACTCCCTGAAACCTGAGGACACAGCCCTGTACTATTGT GCCGCCGATCGGAAGAGCGTGATGAGCATTAGACCAGACT ATTGGGGGCAGGGAACACAGGTGACCGTGAGCAGC(Sequence ID) 4) LCAR-B38M CD8α Hinge Nucleic Acid Sequence ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGT GCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCT GGACTTCGCCTGTGAT(Sequence ID 14) LCAR-B38M CD8α transmembrane nucleic acid sequence ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTC CTTCTCCTGTCACTGGTTATCACCCTTTACTGC (Sequence ID 9) LCAR-B38M CD137 cytoplasmic nucleic acid sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATG TGAACTG (Sequence ID 13) LCAR-B38M CD3ζ cytoplasmic nucleic acid sequence AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGA TGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTC AGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 11)

Claims

1. A method for treating a subject with cancer, a) Extracellular antigen-binding dormant containing a first anti-BCMA binding moiety and a second BCMA binding moiety In and b) Transmembrane domain and c) Chimeric antigen receptor (CAR) polypeptide containing an intracellular signaling domain. This includes administering at least a single dose of cells containing the above to the subject, Selectively, the cells are CAR-T cells. The aforementioned method.

2. The method according to claim 1, wherein the cells are grown in vitro before injection.

3. The cell is a T cell, an NK cell, or an iPSC-NK cell, according to claim 1 or 2. method.

4. The method according to claim 3, wherein the cells are CAR-T cells.

5. The claim states that the cells are NKT cells, iPSC-T cells, or gamma-delta T cells. The method described in item 4.

6. The method according to any one of claims 1 to 5, wherein the cells are heterologous or self.

7. The aforementioned dosage is 4.0 × 10 per kilogram of the mass of the subject. ５ ~1.0 x 10 ６ pieces The method according to any one of claims 1 to 6, comprising the CAR-T cells.

8. The aforementioned dosage is 5.5 × 10 per kilogram of the mass of the subject. ５ ~8 x 10 ５ Before this one The method according to any one of claims 1 to 7, comprising the CAR-T cells.

9. The aforementioned dosage is approximately 7.5 × 10 per kilogram of the mass of the subject. ５ The aforementioned CAR- The method according to any one of claims 1 to 8, comprising T cells.

10. The above dose is 1 × 10 ６ ~1 x 10 ８ The claims 1 to 9, comprising the aforementioned CAR-T cells The method described in any one of the items.

11. The dosage is 2×10 ７ to 8×10 ７ including the CAR-T cells, according to claims 1 to 10 The method described in any one of the items.

12. The aforementioned dose is approximately 5.25 × 10 ７ The following claims, comprising the aforementioned CAR-T cells The method described in either of the above terms.

13. The aforementioned cells amount to 4.0 × 10⁶ cells per kilogram of the mass of the subject. ５ ~1.0 x 10 ６ pieces The dose is administered to produce the aforementioned CAR-T cells, as described in any one of claims 1 to 6. The method.

14. The aforementioned cells amount to 5.5 × 10 per kilogram of the mass of the subject. ５ ~8 x 10 ５ Before this one The following is a drug administered in a dose that produces CAR-T cells, according to any one of claims 1 to 6 and 13. Method of description.

15. The aforementioned cells number approximately 7.5 × 10 per kilogram of the mass of the subject. ５ The aforementioned CAR- Administered in a dose that produces T cells, as described in any one of claims 1 to 6, 13, and 14. Method of loading.

16. The aforementioned cells are 1 × 10 in the subject. ６ ~1 x 10 ８ Having the aforementioned CAR-T cells The method according to any one of claims 1 to 7 and 13 to 15, administered in a dose.

17. The aforementioned cells are 2 × 10 in the subject. ７ ~8 x 10 ７ Having the aforementioned CAR-T cells The method according to any one of claims 1 to 7 and 13 to 16, administered in a dose.

18. The aforementioned cells are approximately 5.25 × 10 in the subject. ７ This results in a number of the aforementioned CAR-T cells. The method according to any one of claims 1 to 7 and 13 to 17, administered in a specified dose.

19. The claim states that the dose of CAR-T cells is administered only once during the course of the treatment. The method described in any one of items 1 to 18.

20. The dose of CAR-T cells is administered intravenously, according to any one of claims 1 to 19. Methods used.

21. The method according to any one of claims 1 to 20, wherein the cancer is multiple myeloma.

22. Claim 21, wherein the multiple myeloma is refractory multiple myeloma or relapsed multiple myeloma. Methods used.

23. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to any one of claims 1 to 22, wherein the ratio to cells is less than 4.

24. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 23, wherein the ratio to cells is less than 2.

25. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 24, wherein the ratio to cells is less than 1.

8.

26. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 25, wherein the ratio to cells is less than 1.

7.

27. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 26, wherein the ratio to cells is less than 1.

6.

28. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 27, wherein the ratio to cells is less than 1.

4.

29. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 28, wherein the ratio to cells is less than 1.

2.

30. CD4+ CAR-T cells in the aforementioned dose of CAR-T cells CD8+ CAR-T cells The method according to claim 29, wherein the ratio to cells is less than 1.

0.

31. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to any one of claims 1 to 30, wherein the ratio to is less than 3.

5.

32. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to any one of claims 1 to 31, wherein the ratio to is less than 2.

0.

33. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to claim 32, wherein the ratio to is less than 1.

2.

34. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to claim 33, wherein the ratio to is less than 0.

8.

35. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to claim 34, wherein the ratio to is less than 0.

6.

36. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to claim 35, wherein the ratio to is less than 0.

4.

37. CD4+ CAR-T cells in Cmax and CD8+ CAR-T cells in Cmax The method according to claim 36, wherein the ratio to is less than 0.

3.

38. The amount of CD4+ CAR-T cells and / or CD8+ CAR-T cells in the subject is The method according to any one of claims 1 to 37, further comprising making a sale.

39. The first BCMA binding portion and / or the second BCMA binding portion are anti-BCMA The method according to any one of claims 1 to 38, wherein sdAb.

40. The first BCMA binding portion is the first anti-BCMA sdAb, and the second BC The method according to claim 39, wherein the MA binding portion is a second anti-BCMA sdAb.

41. The first BCMA bond portion is QVKLEESGGGGLVQAGRSLRLSCAA SEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSV KGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAA Claims 1 to 40, comprising the amino acid sequence DFDSWGQGTQVTVSS (SEQ ID NO: 1) The method described in any one of the items.

42. The first BCMA bond portion is CAGGTCAAACTGGAAGAATCTGGGC GGAGGCCTGGTGCAGGCAGGACGGAGCCTGCGCCTGAGCT GCGCAGCATCCGAGCACACCTTCAGCTCCCACGTGATGGG CTGGTTTCGGCAGGCCCCAGGCAAGGAGAGAGAGAGCGTG GCCGTGATCGGCTGGAGGGACATCTCCACATCTTACGCCG ATTCCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACGC CAAGAAGACACTGTATCTGCAGATGAACAGCCTGAAGCCCC GAGGACACCGCCGTGTACTATTGCGCAGCAAGGAGAATCG ACGCAGCAGACTTTGATTCCTGGGGCCAGGGCACCCAGGT The nucleic acid sequence GACAGTGTCTAGC (SEQ ID NO: 2) encodes the polypeptide The method according to any one of claims 1 to 40, including D.

43. The second BCMA bond portion is EVQLVESGGGLVQAGGSLRLSCAA SGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGR FTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIR Claims 1 to 42, comprising the amino acid sequence PDYWGQGTQVTVSS (SEQ ID NO: 3) The method described in any one of the items.

44. The second BCMA bond portion is GAGGTGCAGCTGGTGAGAGCGGA GGCGGCCTGGTGCAGGCCGGAGGCTCTCTGAGGCTGAGCT GTGCAGCATCCGGAAGAACCTTCACAATGGGCTGGTTTAG GCAGGCACCAGGAAAGGAGAGGGGAGTTCGTGGCAGCAATC AGCCTGTCCCCCTACCCTGGCCCTACTATGCCGAGAGGCGTGA AGGGCAGGTTTACCATCTCCCGCGATAACGCCAAGAATAC AGTGGTGCTGCAGATGAACTCCCTGAAACCTGAGGACACA GCCCTGTACTATTGTGCCGCCGATCGGAAGAGCGTGATGA GCATTAGACCAGACTATTGGGGGCAGGGGAACACAGGTGAC Contains a polypeptide encoded by the nucleic acid sequence CGTGAGCAGC (SEQ ID NO: 4) The method according to any one of claims 1 to 43.

45. The first BCMA-binding portion and the second BCMA-binding portion connect to the peptide linker. The method according to any one of claims 1 to 44, wherein the elements are connected to each other via a bridging device.

46. Claim 4, wherein the peptide linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 5). The method described in 5.

47. The CAR polypeptide has a signal peptide located at the N-terminus of the polypeptide. The method according to any one of claims 1 to 46.

48. The method according to claim 47, wherein the signal peptide is derived from CD8α.

49. The signal peptide, The amino acid sequence of MALPVTALLLPLAALLLHAARP (SEQ ID NO: 6) is included. The method according to claim 48.

50. The signal peptide, ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCT The nucleic acid sequence of CTGCTGCCTGCACGCCTGCCCCT (SEQ ID NO: 7) The method according to claim 48, comprising a coded polypeptide.

51. The aforementioned transmembrane domain is IYIWAPLAGTCGVLLLLSLVITLYC (sequence number) The method according to any one of claims 1 to 48, comprising the amino acid sequence of (8).

52. The transmembrane domain is ACTTACATCTGGGCCCCCTTGGCCGGGA CTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTTA Claim 1 comprises a polypeptide encoded by the nucleic acid sequence CTGC (SEQ ID NO: 9). The method described in any one of items ~48.

53. The aforementioned intracellular signaling domain is the major intracellular signaling domain of immune effector cells. The method according to any one of claims 1 to 52, including the domain.

54. The intracellular signaling domain is derived from CD3ζ, any one of claims 1 to 52. The method described in item 1.

55. The intracellular signaling domain is one or more co-stimulatory signaling domains The method according to any one of claims 1 to 54, including the method described in any one of claims 1 to 54.

56. The intracellular signaling domain is RVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD The method according to claim 55, comprising the amino acid sequence of ALHMQALPPR (SEQ ID NO: 10). 。

57. The intracellular signaling domain is AGAGTGAAGTTCAGCAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCCAATCTAGGACGAAGAGAGGAGTACGATGTT TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA ACTGCAGAAAGATAAGATGGCGGAGGCCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGGCAAGGGGCACGATG GCCTTTACCAGGGTCTCAGTACAGCCACCAGGACACCTA CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (sequence The method according to claim 55, comprising a polypeptide encoded by the nucleic acid sequence of number 11). Law.

58. The intracellular signaling domain is KRGRKKLLYIFKQPFMRPVQTT The amino acid sequence QEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12) is included. The method described in any one of the requests 55 to 57.

59. The intracellular signaling domain is AAACGGGGCAGAAAGAACTCC TGTATATATTCAAACAACCATTTATGAGACCAGTACAAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAA The nucleic acid sequence GAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 13) The method according to any one of claims 55 to 57, comprising a polypeptide encoded by

60. The CAR polypeptide has a C-terminus of the extracellular antigen-binding domain and a transmembrane domain. Any one of claims 1 to 59 further comprises a hinge domain located between the N-terminus and the Methods used.

61. The aforementioned hinge domain is TTTTPAPRPPPAPTIASQPLSLRPEACR Claim 6, comprising the amino acid sequence of PAAGGAVHTRGLDFACD (SEQ ID NO: 15) The method described in 0.

62. The hinge domain is ACCACGACGCCCAGGCGCGCACCACAA CACCGGCGCCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC Nucleic acid sequence of ACGAGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 14) The method according to claim 60, comprising a polypeptide encoded by

63. The method according to any one of claims 1 to 62, wherein the T cells are autologous T cells.

64. The method according to any one of claims 1 to 62, wherein the T cells are allogeneic T cells.

65. The method according to any one of claims 1 to 64, wherein the subject is a human.

Images