Antigen binding polypeptides

Abstract

The present disclosure provides compositions comprising antigen-binding polypeptides. More particularly, the present disclosure relates to polypeptides comprising an antibody or antigen-binding fragment thereof, nucleic acids encoding the polypeptides, and vectors for expressing the polypeptides.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 540,332, filed September 25, 2023, and U.S. Provisional Application No. 63 / 618,880, filed January 8, 2024, pursuant to 35 USC § 119(e), which are incorporated herein by reference in their entirety.

[0003] Declaration of sequence list

[0004] The sequence list related to this application is provided in XML format, not in a paper copy, and is incorporated herein by reference. The XML file containing the sequence list is named KELO-011-WO1_ST26.xml. The XML file is 1176 KB in size, created on September 20, 2024, and submitted electronically through the Patent Center, simultaneously with the submission of the specification. Technical Field

[0005] This disclosure relates to antigen-binding polypeptides. More specifically, this disclosure relates to polypeptides comprising antibodies or antigen-binding fragments thereof, nucleic acids encoding said polypeptides, and vectors for expressing said polypeptides. Background Technology

[0006] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17). BCMA is normally expressed in mature B lymphocytes and plasma cells. BCMA expression has also been detected in various lymphomas and multiple myeloma. Multiple myeloma is an incurable malignant plasma cell tumor originating in the bone marrow.

[0007] Multiple myeloma is the second most common hematologic malignancy after non-lymphomas. In 2020, approximately 176,404 people worldwide were diagnosed with multiple myeloma, and about 117,077 patients died from the disease. In 2023, in the United States alone, an estimated 35,730 people will be diagnosed with multiple myeloma, and an estimated 12,590 patients with multiple myeloma will die from the disease or related complications. In the United States, the 5-year relative survival rate for multiple myeloma is only about 58%.

[0008] Multiple myeloma can initially be treated with autologous stem cell transplantation (ASCT) and / or various drug combinations (e.g., proteasome inhibitors, including bortezomib, carfilzomib, and ixazomib; immunomodulatory drugs (IMiDs), including pomalidomide, lenalidomide, and thalidomide; and corticosteroids, such as dexamethasone), but patients eventually relapse, and the disease becomes difficult to treat. Subsequent treatment options include monoclonal antibodies, bispecific antibodies (e.g., BiTE), antibody-drug conjugates, and ultimately chimeric antigen receptor T-cell therapy.

[0009] Autologous ex vivo chimeric antigen receptor (CAR) T-cell therapy is emerging as a late-stage treatment option for patients with multiple myeloma. Despite the promise of these ex vivo CAR T-cell therapies, they have not yet reached their full potential due to long and expensive drug development cycles, limited availability to only a few treatment centers with the expertise to provide them, their association with a high incidence of cytokine release syndrome, and the fact that most patients ultimately relapse and die from the disease. Significant unmet needs remain for more affordable, readily available, and effective treatments for patients with multiple myeloma. Summary of the Invention

[0010] This disclosure generally relates in part to antibodies against B cell maturation antigen (BCMA) and antigen-binding fragments thereof, polypeptides comprising anti-BCMA antibodies or antigen-binding fragments thereof, bispecific antibodies comprising anti-BCMA antibodies or antigen-binding fragments thereof and anti-CD3 antibodies, immunoconjugates comprising anti-BCMA antibody drugs linked to cytotoxic agents, anti-BCMA chimeric antigen receptors, polynucleotides encoding said polypeptides, vectors for expressing said polynucleotides, and compositions comprising the above substances.

[0011] In various embodiments, this disclosure partially contemplates antibodies or antigen-binding fragments thereof comprising: (a) heavy chain variable regions (VH) of CDRH1, CDRH2, and CDRH3 containing the antibodies or antigen-binding fragments thereof shown in Table 1; peptide linkers; and light chain variable regions (VL) of CDRL1, CDRL2, and CDRL3 containing the antibodies or antigen-binding fragments thereof shown in Table 1; or (b) VHH domains of CDRH1, CDRH2, and CDRH3 containing the antibodies or antigen-binding fragments thereof shown in Table 1.

[0012] In specific embodiments: (a) a VH comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 12, 13, and 14; and a VL comprising CDRL1, CDRL2, and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 16, 17, and 18; (b) a VH comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 22, 23, and 24; and a VL comprising CDRL1, CDRL2, and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 26, 27, and 28; (c) a VH comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 32, 33, and 34; and a VL comprising CDRL1, CDRL2, and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 36, 37, and 38; (d) a VL comprising containing the amino acid sequences shown in SEQ ID NO: VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 42, 43, and 44; and VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 46, 47, and 48; (e) VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 52, 53, and 54; and VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 56, 57, and 58; (f) VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 62, 63, and 64; and VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 66, 67, and 68; (g) VL containing SEQ ID NO: VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 72, 73, and 74; and VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 76, 77, and 78; (h) VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 82, 83, and 84; and VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 86, 87, and 88; (i) VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 92, 93, and 94;and VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 96, 97, and 98; (j) VHH domain containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 102, 103, and 104; (k) VHH domain containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 106, 107, and 108; (l) VHH domain containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 110, 111, and 112; (m) VHH domain containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 114, 115, and 116; (n) VHH domain containing SEQ ID NO: The following are examples of compounds containing VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 118, 119, and 120: (o) containing VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 122, 123, and 124; (p) containing VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 126, 127, and 128; (q) containing VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 130, 131, and 132; (r) containing VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 134, 135, and 136; (s) containing VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 134, 135, and 136. The VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 138, 139, and 140; or (t) comprising the VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 142, 143, and 144. In a specific embodiment, VH comprises CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64, while VL comprises CDRL1, CDRL2, and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 66, 67, and 68.

[0013] In some embodiments: (a) VH contains the amino acid sequence shown in SEQ ID NO: 11, and VL contains the amino acid sequence shown in SEQ ID NO: 15; (b) VH contains the amino acid sequence shown in SEQ ID NO: 21, and VL contains the amino acid sequence shown in SEQ ID NO: 25; (c) VH contains the amino acid sequence shown in SEQ ID NO: 31, and VL contains the amino acid sequence shown in SEQ ID NO: 35; (d) VH contains the amino acid sequence shown in SEQ ID NO: 41, and VL contains the amino acid sequence shown in SEQ ID NO: 45; (e) VH contains the amino acid sequence shown in SEQ ID NO: 51, and VL contains the amino acid sequence shown in SEQ ID NO: 55; (f) VH contains the amino acid sequence shown in SEQ ID NO: 61, and VL contains the amino acid sequence shown in SEQ ID NO: 65; (g) VH contains the amino acid sequence shown in SEQ ID NO: 71, and VL contains the amino acid sequence shown in SEQ ID NO: 75; (h) VH contains the amino acid sequence shown in SEQ ID NO: 11, and VL contains the amino acid sequence shown in SEQ ID NO: 15. The amino acid sequence shown in SEQ ID NO: 81, wherein VL comprises the amino acid sequence shown in SEQ ID NO: 85; (i) VH comprises the amino acid sequence shown in SEQ ID NO: 91, and VL comprises the amino acid sequence shown in SEQ ID NO: 95; or (a) the VHH domain comprises any one of the amino acid sequences shown in SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141. In a specific embodiment, VH comprises the amino acid sequence shown in SEQ ID NO: 61, and VL comprises the amino acid sequence shown in SEQ ID NO: 65.

[0014] In some embodiments, the peptide linker is selected from: TGEKP (SEQ ID NO: 2); (GGGGS) n , wherein n=1, 2, 3, 4 or 5 (SEQ ID NO: 3, 976-979); EGKSSGSGSESKVD (SEQ ID NO: 4); KESGSSVSSEQLAQFRSLD (SEQ ID NO: 5); LRQRDGERP (SEQ ID NO: 6); LRQKDGGGSERP (SEQ ID NO: 7); LRQKD(GGGS)ERP (SEQ ID NO: 8), GEGTSTGSGGSGGSGGAD (SEQ ID NO: 9) and GSTGSSGKPGSGEGSTKG (SEQ ID NO: 10).

[0015] In a specific embodiment, the antibody or its antigen-binding fragment comprises any of the following amino acid sequences: SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141, preferably SEQ ID NO: 69 or 70.

[0016] In various embodiments, this disclosure includes consideration of bispecific antibodies comprising the antibodies or antigen-binding fragments thereof considered herein.

[0017] In some implementations, the bispecific antibody also includes an anti-CD3 antibody that binds to CD3δ, CD3ε, CD3γ, or CD3ζ.

[0018] In various embodiments, this disclosure includes conjugates comprising antibodies or antigen-binding fragments thereof considered herein.

[0019] In some implementations, the antigen or its antigen-binding fragment is conjugated to a cytotoxic agent.

[0020] In the specific implementation scheme: (a) the cytotoxic agent is selected from the following toxins: saponins, diphtheria toxin, Pseudomonas exotoxin A, ricin A chain derivatives, small molecule toxins, and combinations thereof; (b) the cytotoxic agent is selected from the following radioisotopes: 131I, 90Y, 177Lu, 188Re, 67Cu, 213Bi, 211At, and 227Ac; (c) the cytotoxic agent is selected from the following RNA polymerase II inhibitors and / or RNA polymerase III inhibitors: Amatoxin, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanic acid, and any functional fragments, derivatives, or analogs thereof; or (d) cytotoxic agents are selected from the group consisting of anti-microtubule agents, DNA cross-linking agents, DNA alkylating agents, and mitotic disruptors.

[0021] In various embodiments, this disclosure includes a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof as considered herein; a spacer domain; a transmembrane domain; and one or more intracellular signal transduction domains.

[0022] In some embodiments, the spacer domain includes a hinge domain selected from the following, or a fragment thereof: CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, and IgG4 hinge.

[0023] In a specific implementation, the spacer domain comprises an amino acid sequence shown in any one of SEQ ID NO: 145, 146, 147, 148, 149 and 150 or an amino acid sequence that is at least 95% identical to it.

[0024] In some embodiments, the transmembrane domain is isolated from or derived from a polypeptide selected from: the α, β, γ or δ chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1).

[0025] In a specific implementation, the transmembrane domain comprises the amino acid sequence shown in any one of SEQ ID NO: 151, 152, 153, 154, 155, 156 and 157 or an amino acid sequence that is at least 95% identical to it.

[0026] In some embodiments, one or more intracellular signal transduction domains comprise major signal transduction domains isolated from or derived from a polypeptide selected from the following: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0027] In some implementations, one or more intracellular signal transduction domains include a primary signal transduction domain separated from CD3ζ.

[0028] In some embodiments, the primary signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to it.

[0029] In a specific implementation, one or more intracellular signal transduction domains comprise co-stimulatory signal transduction domains isolated from or derived from a selection of peptides including: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

[0030] In some embodiments, one or more intracellular signal transduction domains include a co-stimulatory signal transduction domain, said co-stimulatory signal transduction domain comprising an amino acid sequence shown in any one of SEQ ID NO: 159, 160, 161, 162, 163 and 164 or an amino acid sequence that is at least 95% identical to it.

[0031] In various embodiments, this disclosure partially contemplates a CAR comprising: an antibody or an antigen-binding fragment thereof comprising an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, 90, 101, or 117; a spacer domain comprising an amino acid sequence shown in any one of SEQ ID NO: 145, 146, and 148, or an amino acid sequence at least 95% identical thereto; a transmembrane domain comprising an amino acid sequence shown in SEQ ID NO: 151 or 153; and one or more intracellular signal transduction domains comprising a co-stimulatory signal transduction domain comprising an amino acid sequence shown in any one of SEQ ID NO: 159, 160, and 162, or an amino acid sequence at least 95% identical thereto; and further comprising a major signal transduction domain comprising an amino acid sequence shown in SEQ ID NO: 158, or an amino acid sequence at least 95% identical thereto.

[0032] In various embodiments, this disclosure partially contemplates CARs comprising the amino acid sequences shown in any of SEQ ID NO: 165-860.

[0033] In various embodiments, this disclosure partially contemplates a CAR comprising any one of the amino acid sequences shown in SEQ ID NO: 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, and 283.

[0034] In various embodiments, this disclosure partially contemplates CARs comprising any one of the amino acid sequences shown in SEQ ID NO: 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, and 380.

[0035] In various embodiments, this disclosure partially contemplates a CAR comprising any one of the amino acid sequences shown in SEQ ID NO: 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, and 452. In a specific embodiment, a CAR comprises the amino acid sequence shown in SEQ ID NO: 429 or an amino acid sequence that is 95% identical thereto.

[0036] In various embodiments, this disclosure partially contemplates CARs comprising the amino acid sequences shown in any one of SEQ ID NO: 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, and 548.

[0037] In various embodiments, this disclosure partially contemplates CARs comprising the amino acid sequences shown in any one of SEQ ID NO: 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, and 620.

[0038] In various embodiments, this disclosure partially contemplates CARs comprising the amino acid sequences shown in any one of SEQ ID NO: 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, and 716.

[0039] In some implementations, the CAR also includes a signal peptide.

[0040] In a specific implementation, the signal peptide comprises any one of the amino acid sequences shown in SEQ ID NO: 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872 and 873.

[0041] In a specific implementation, the polynucleotide encoding CAR comprises any one of the polynucleotide sequences shown in SEQ ID NO: 905-924.

[0042] In a specific implementation, the polynucleotide encoding the signal peptide and CAR comprises any one of the polynucleotide sequences shown in SEQ ID NO: 925-944.

[0043] In various embodiments, this disclosure includes consideration of polynucleotides encoding antibodies or antigen-binding fragments thereof, bispecific antibodies, antibody conjugates, or CARs considered herein.

[0044] In various embodiments, this disclosure partially contemplates polynucleotides encoding or containing promoters operatively linked to any of the polynucleotides shown in SEQ ID NO: 905-944.

[0045] In some embodiments, the promoter comprises any one of SEQ ID NO: 948, 949, 950, 951, 952 and 953, preferably the polynucleotide sequence shown in SEQ ID NO: 949.

[0046] In specific implementations, the polynucleotide also includes a post-transcriptional response element.

[0047] In some implementations, the posttranscriptional response element comprises a polynucleotide sequence shown in any one of SEQ ID NO: 945, 946, and 947.

[0048] In various embodiments, this disclosure partially contemplates DNA comprising the polynucleotide sequence shown in any one of SEQ ID NO: 945, 946 and 947.

[0049] In various embodiments, this disclosure partially considers RNA encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 945, 946 and 947.

[0050] In various embodiments, this disclosure partially contemplates vectors comprising the polynucleotide sequences shown in any one of SEQ ID NO: 945, 946 and 947.

[0051] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters containing the sequence shown in SEQ ID NO: 950, the promoters being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0052] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters containing the sequence shown in SEQ ID NO: 950, the promoters being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141, the antibody or the antigen-binding fragment thereof, and optionally a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947.

[0053] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters containing the sequence shown in SEQ ID NO: 950, the promoters being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor containing any one of the amino acids shown in SEQ ID NO: 165-860, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0054] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters comprising the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising an amino acid represented by any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element represented by any one of SEQ ID NO: 945-947.

[0055] In various embodiments, this disclosure partially contemplates vectors that encode or contain promoters comprising the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924, and optionally comprising a polynucleotide containing a posttranscriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0056] In a specific implementation, the vector encoding or containing a promoter comprises the sequence shown in SEQ ID NO: 950, said sequence being operatively linked to a polynucleotide comprising a polynucleotide sequence comprising any one of SEQ ID NO: 925-944, and optionally a polynucleotide comprising a posttranscriptional regulatory element comprising any one of SEQ ID NO: 945-947.

[0057] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters containing the sequence shown in SEQ ID NO: 949, the promoters being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0058] In various embodiments, this disclosure partially contemplates vectors encoding or comprising a promoter comprising the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141, and optionally comprising a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947.

[0059] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters containing the sequence shown in SEQ ID NO: 949, the promoters being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor containing any one of the amino acids shown in SEQ ID NO: 165-860, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0060] In various embodiments, this disclosure partially contemplates vectors encoding or comprising promoters containing the sequence shown in SEQ ID NO: 949, the promoters being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising any one of the amino acids shown in SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837 (preferably SEQ ID NO: 429), and optionally comprising a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0061] In various embodiments, this disclosure partially contemplates vectors that encode or contain promoters comprising the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide comprising the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924 (preferably SEQ ID NO: 910), and optionally a polynucleotide containing a posttranscriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0062] In some embodiments, the vector encoding or containing a promoter comprises the sequence shown in SEQ ID NO: 949, which is operatively linked to a polynucleotide comprising a polynucleotide sequence comprising any one of SEQ ID NO: 925-944 (preferably SEQ ID NO: 930), and optionally a polynucleotide comprising a posttranscriptional regulatory element comprising any one of SEQ ID NO: 945-947.

[0063] In some implementations, the carrier is an expression carrier.

[0064] In specific implementation schemes, the vector is a transfer plasmid or a viral vector.

[0065] In some implementations, the vector is a plasmid.

[0066] In a specific implementation plan, the vector is a viral vector selected from adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, parvovirus vectors, rhabdovirus vectors, vesicular virus vectors, paramyxovirus vectors, measles virus vectors, hennipa virus vectors, alphavirus vectors, flavivirus vectors, retrovirus vectors, and lentivirus vectors (LVV).

[0067] In some implementations, the lentiviral vector is genome-engineered or derived from a lentivirus selected from: HIV (HIV type 1 or HIV type 2); Vesner-Medy virus (VMV); Capillary Arthritis-Encephalitis Virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV).

[0068] In various embodiments, this disclosure considers lentiviral vectors comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding or containing a promoter operatively linked to the polynucleotide sequence described in SEQ ID NO: 904 and any one of SEQ ID NO: 905-924 (preferably SEQ ID NO: 910); an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and a poly(A) tail.

[0069] In various embodiments, this disclosure considers lentiviral vectors comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding or containing a promoter operatively linked to the polynucleotide sequence shown in any one of SEQ ID NO: 925-944 (preferably SEQ ID NO: 930); an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and a poly(A) tail.

[0070] In various embodiments, this disclosure contemplates an RNA comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding a promoter operatively linked to the polynucleotide sequence described in SEQ ID NO: 904 and any one of SEQ ID NO: 905-924 (preferably SEQ ID NO: 910); an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and an optional poly(A) tail.

[0071] In various embodiments, this disclosure contemplates an RNA comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding a promoter operatively linked to the polynucleotide sequence shown in any one of SEQ ID NO: 925-944 (preferably SEQ ID NO: 930); an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and an optional poly(A) tail.

[0072] In various embodiments, this disclosure considers recombinant lentiviruses comprising one or more copies of the lentiviral vector or RNA considered herein.

[0073] In various embodiments, this disclosure partially contemplates compositions comprising an antibody or an antigen-binding fragment thereof, a bispecific antibody, an antibody conjugate, a CAR, a polynucleotide, a vector, RNA, or a recombinant lentivirus as considered herein. Attached Figure Description

[0074] Figure 1This is a schematic diagram of a vector that encodes a promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR) and an optional post-transcriptional response element (PRE) operatively linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

[0075] Figure 2A The Jurkat cell titer of the recombinant lentivirus is shown. The recombinant lentivirus comprises: a viral envelope expressing mutant vesicular stomatitis Indiana virus envelope glycoprotein G (VSIV-G) and a nonviral membrane-binding kinetic polypeptide that binds CD3; and a lentiviral vector encoding an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated).

[0076] Figure 2B The expression of anti-BCMA CAR on PBMCs transduced with recombinant lentiviral particles is shown. The recombinant lentiviral particles comprise: a viral envelope expressing the mutant VSIV-G and a nonviral membrane-binding kinetic peptide that binds to CD3; and a lentiviral vector encoding an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated).

[0077] Figure 2C The vector copy number (VCN) in PBMCs transduced with recombinant lentiviral particles is shown. The recombinant lentiviral particles comprise: a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide that binds to CD3; and a lentiviral vector encoding an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated).

[0078] Figure 2DAnti-BCMA CAR activity was demonstrated, measured by the amount of IFNγ produced in a co-culture assay. PBMCs transduced with recombinant lentiviral particles were co-cultured for 24 hours with Daudi cells (low BCMA expression) or RPMI-8226 cells (high BCMA expression). The recombinant lentiviral particles comprised: a viral envelope expressing a mutant VSIV-G and a non-viral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector encoding an MNDU3 promoter operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operably linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated). After 24 hours, IFNγ levels in the co-culture supernatant were measured and plotted as a function of the percentage of anti-BCMA CAR-positive cells in the co-culture.

[0079] Figure 3A The functional titers of recombinant lentiviruses in Jurkat cells are shown. These recombinant lentiviruses comprise a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide that binds to CD3; and a lentiviral vector encoding an MNDU3 promoter, SFFV promoter, or EF1α promoter operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR (six anti-BCMA CARs were evaluated), without PRE, or with a wild-type or mutant WPRE operably linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

[0080] Figure 3B The percentage of transduced PBMCs showing VCN as a function of the percentage of PBMCs expressing anti-BCMA CAR is displayed. Human PBMCs were transduced with a recombinant lentivirus comprising a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; and a lentiviral vector encoding an MNDU3 promoter, SFFV promoter, or EF1α promoter operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR (six anti-BCMA CARs were evaluated), either without PRE or with a wild-type or mutant WPRE operably linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

[0081] Figure 3C The amount of IFNγ secreted by PBMCs expressing anti-BCMACAR after 24 hours of co-culturing with RPMI-8226 cells (cells expressing BCMA) was shown as a function of the percentage of CAR-expressing cells in the co-culture.

[0082] Figure 3DThe amount of IL-2 secreted by PBMCs expressing anti-BCMACAR after 24 hours of co-culturing with RPMI-8226 cells (cells expressing BCMA) was shown as a function of the percentage of CAR-expressing cells in the co-culture.

[0083] Figure 3E The amount of IFNγ secreted by PBMCs expressing the anti-BCMA CAR in the absence of target cells is shown. Human PBMCs were transduced with a recombinant lentivirus comprising: a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding an anti-BCMA CAR (six anti-BCMA CARs were evaluated).

[0084] Figure 3F Off-target transduction levels in BCMA-expressing cells (RPMI-8226 and KMS-11) with recombinant lentiviruses comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding anti-BCMA CAR (six anti-BCMA CARs were evaluated). In cells transduced with a recombinant lentivirus encoding GFP instead of the anti-BCMA CAR, transduction was normalized to VCN.

[0085] Figure 4A Results are shown in an in vivo Daudi mouse model. Mice were administered a recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; a lentiviral vector encoding an MNDU3 promoter operably linked to a CD8α signal peptide and an anti-BCMA CAR polynucleotide (four anti-BCMA CARs were evaluated), and a wild-type WPRE operably linked to the 3' end of the anti-BCMA CAR polynucleotide.

[0086] Figure 4BResults from an in vivo Daudi mouse model are shown. Mice were administered recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding an anti-BCMA CAR (five different anti-BCMA CARs were evaluated).

[0087] Figure 4C Results from an in vivo Daudi mouse model are shown. Mice were administered recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding an anti-BCMA CAR (four different anti-BCMA CARs were evaluated).

[0088] Figure 4D Results from the in vivo RPMI-8226 mouse model are shown. Mice were administered recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: an MNDU3 promoter with a mutant WPRE, or an EF1α promoter with no WPRE, encoding an anti-BCMA CAR (two different anti-BCMA CARs were evaluated).

[0089] Figure 4E Results from an in vivo RPMI-8226 mouse model are shown. Mice were administered three doses (1.25 x 10⁻⁶). 7 IU, 5.0 x 10 7 IU or 1.25 x 10 8 The recombinant lentivirus, ex vivo CAR T cells, or vector comprising (IU) a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; and a lentiviral vector comprising an EF1α promoter operatively linked to a polynucleotide encoding anti-BCMA CAR, without PRE.

[0090] Figure 4F Results from an in vivo RPMI-8226 mouse model are shown. Mice were administered two doses (5.0 x 10⁻⁶).7 IU or 1.25 x 10 8 The recombinant lentivirus, ex vivo CAR T cells, or vector comprising (IU) a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; and a lentiviral vector comprising an EF1α promoter operatively linked to a polynucleotide encoding anti-BCMA CAR, without PRE.

[0091] Figure 4G Results from an in vivo Daudi mouse model are shown. Mice were administered a recombinant lentivirus, in vitro CAR T cells, or a vector comprising a viral envelope expressing a mutant VSIV-G and a non-viral membrane-binding kinetic peptide binding to CD3, and a lentiviral vector encoding an anti-BCMA CAR, and comprising one of the following lentiviral vector structures: an MNDU3 promoter and a mutant WPRE, or an MNDU3 promoter and a mutant WPRE (1.25 x 10⁻⁶). 8 IU), or EF1α promoter and no WPRE (5.6 x 10) 7 IU).

[0092] Figure 5A Results from an in vivo lentivirus study of the Daudi mouse model are shown. Mice were administered a vector control or a recombinant lentivirus comprising a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3, and a lentiviral vector encoding one of three anti-BCMA CARs or a GFP control.

[0093] Figure 5B Results from a Daudi mouse model studied using CAR T cells manufactured in vitro are shown. Mice were administered a vector control, untransduced PBMCS, or PBMs transduced with a recombinant lentivirus containing a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic peptide that binds CD3, and a lentiviral vector encoding one of three anti-BCMA CARs.

[0094] Brief description of sequence identifiers

[0095] SEQ ID NO: 1 lists the amino acid sequence of the B cell maturation antigen (BCMA) polypeptide.

[0096] SEQ ID NO: 2-10 and 976-979 list the amino acid sequences of the polypeptide linkers.

[0097] SEQ ID NO: 11-144 lists the amino acid sequence of the antibody.

[0098] SEQ ID NO: 145-150 lists the amino acid sequences of the spacer domain.

[0099] SEQ ID NO: 151-157 lists the amino acid sequences of the transmembrane domain.

[0100] SEQ ID NO: 158-164 lists the amino acid sequences of intracellular signal transduction domains.

[0101] SEQ ID NO: 165-860 lists the amino acid sequence of the chimeric antigen receptor (CAR).

[0102] SEQ ID NO: 861-873 lists the amino acid sequence of the signal peptide.

[0103] SEQ ID NO: 874-893 lists the nucleic acid sequences encoding the antibodies.

[0104] SEQ ID NO: 894-897 lists the nucleic acid sequences encoding the spacer domain.

[0105] SEQ ID NO: 898-899 lists nucleic acid sequences encoding transmembrane domains.

[0106] SEQ ID NO: 900-903 lists nucleic acid sequences encoding intracellular signal transduction domains.

[0107] SEQ ID NO: 904 lists the nucleic acid sequence encoding the signal peptide.

[0108] SEQ ID NO: 905-924 lists nucleic acid sequences encoding chimeric antigen receptors (CARs) that do not contain a signal peptide.

[0109] SEQ ID NO: 925-944 lists the nucleic acid sequences encoding CARs containing signal peptides.

[0110] SEQ ID NO: 945-947 lists the nucleic acid sequences of post-transcriptional response elements.

[0111] SEQ ID NO: 948-953 lists the nucleic acid sequence of the promoter.

[0112] SEQ ID NO: 954-955 lists the amino acid sequences of anti-BCMA CAR.

[0113] SEQ ID NO: 956-975 lists the amino acid sequence of the viral self-cleavage peptide.

[0114] In the aforementioned sequence, X (if present) refers to any amino acid, a specific set of amino acids, or the absence of an amino acid. Detailed Implementation

[0115] A. Overview

[0116] Chimeric antigen receptors (CARs) are used to redirect immune effector cells to target cells. Typically, immune effector cells are harvested from a patient, modified in vitro with a vector to express a CAR, and then reinfused into the patient. In the patient, the CAR-expressing immune effector cells seek out and destroy target cells, such as cancer cells.

[0117] Careful vector design and consideration of CAR structure have jointly contributed to effective CAR-based therapies. Vector design considerations include, but are not limited to, the choice of vector type (e.g., viral or non-viral vector); promoter selection; selection of post-transcriptional regulatory elements; and so on. CARs contain several components, including, but not limited to, a target antigen-binding moiety, such as a ligand, antibody, or its antigen-binding fragment; a spacer domain that positions the target-binding domain at an appropriate distance from the surface of immune effector cells; a transmembrane domain that anchors the CAR to immune effector cells; and one or more intracellular signaling domains that transduce extracellular signals into an intracellular signaling cascade that provides a durable and effective immune response. Excessive CAR expression or activity may lead to persistent signaling (activation of immune effector cells in the absence of target cells), while insufficient CAR expression or activity may lead to ineffective recognition and destruction of target cells.

[0118] Recently, ex vivo CAR T-cell therapy targeting B-cell maturation antigen (BCMA) has been used to treat relapsed and refractory multiple myeloma. Although many patients with multiple myeloma treated with ex vivo anti-BCMA CAR T-cell therapy experience partial or complete remission, the majority relapse and die from the disease. There is a significant unmet need for durable, one-time, and potentially curative treatment for multiple myeloma.

[0119] This disclosure provides solutions to the aforementioned and other challenges in the field of treating multiple myeloma using anti-BCMA-binding protein.

[0120] This disclosure generally relates in part to anti-BCMA binding proteins comprising an antibody against BCMA or an antigen-binding fragment thereof. In specific embodiments, the anti-BCMA binding protein is an anti-BCMA antibody or an antigen-binding fragment thereof; a polypeptide comprising an anti-BCMA antibody or an antigen-binding fragment thereof; a bispecific antibody comprising an anti-BCMA antibody or an antigen-binding fragment thereof and an anti-CD3 antibody; an immunoconjugate comprising an anti-BCMA antibody drug linked to a cytotoxic agent; or an anti-BCMA chimeric antigen receptor.

[0121] This disclosure also relates in part to polynucleotides encoding polypeptides, vectors for expressing polynucleotides, and compositions comprising the foregoing.

[0122] In a specific implementation, the chimeric antigen receptor comprises one or more anti-BCMA antibodies or their antigen-binding fragments. Compared to existing anti-BCMA CARs, anti-BCMA CARs offer several advantages, including but not limited to: reduced immunogenicity because the CAR component is derived from human proteins; improved cytokine profile, including increased expression of interferon-γ (IFNγ) and interleukin-2 (IL-2) in the presence of BCMA-expressing target cells; low or absent persistent signaling (antigen-independent signaling); and improved efficacy in mouse models compared to existing CARs.

[0123] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, liposome transfection), enzymatic reactions, purification, and related techniques and procedures can generally be performed according to the descriptions in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology cited and discussed in this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual , 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology , Greene Pub. Associates and Wiley-Interscience (2002); Glover, DNA Cloning: A Practical Approach Volumes I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and ApplicationsJulie Logan, edited by Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes , (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid the Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, ed., 3rd edition, 2010 Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); the treatise, Methods in Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (QEColigan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991); Annual Review of Immunology ; and journals such as Advances in Immunology Thematic papers in the journal.

[0124] B. Definition

[0125] Before elaborating on this disclosure in more detail, providing definitions for certain terms that will be used herein may help in understanding this disclosure.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in practice or testing of particular embodiments, this document describes embodiments of preferred compositions, methods, and materials. For the purposes of this disclosure, the following terms are defined below.

[0127] The articles “a” and “the” are used in this document to refer to one or more of the grammatical objects of the article “the” (i.e., at least one or more). By way of example, “a element” means one element or one or more elements.

[0128] The use of substitution (e.g., "or") should be understood to mean one, both, or any combination of the listed substitutions.

[0129] The term “and / or” should be understood to mean one or both of the alternatives.

[0130] As used herein, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length. In one embodiment, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that is ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length.

[0131] In one implementation, the range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each value covered by the range. For example, in a non-limiting and illustrative embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0132] As used herein, the term “substantially” means that the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher of a reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length. In one embodiment, “substantially identical” means that the effect (e.g., physiological effect) produced by the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length is approximately the same as that of the reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length.

[0133] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of one or a group of said steps or elements, but not to exclude any other steps or elements or any other group of steps or elements. “Comprising” means including and limited to anything following the phrase “comprising.” Therefore, the phrase “comprising” indicates that the listed element is required or mandatory, and that no other element may be present. The phrase “substantially comprising” means including any element listed following the phrase, and without interfering with or contributing to other elements specified in the disclosure for the activity or function of the listed element. Therefore, the phrase “substantially comprising” indicates that the listed element is required or mandatory, but that no other elements substantially affect the activity or function of the listed element.

[0134] Throughout this specification, references to “an embodiment,” “an embodiment,” “a specific embodiment,” “a related embodiment,” “a particular embodiment,” “another embodiment,” or “another embodiment,” or combinations thereof, mean that a specific feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment. Therefore, the appearance of these phrases throughout this specification does not necessarily refer to the same embodiment. Furthermore, the specific feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive description of a feature in one embodiment serves as the basis for excluding said feature in a specific embodiment.

[0135] The terms "binding domain," "extracellular binding domain," and "extracellular antigen-binding domain" are used interchangeably and refer to the domain that enables a chimeric antigen receptor (CAR) to specifically bind to a target antigen. Binding domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0136] A "spacer domain" refers to a polypeptide domain or amino acid sequence in a chimeric antigen receptor that functions to position the antigen-binding domain away from the surface of immune effector cells to achieve proper cell / cell contact, antigen binding, and activation. In specific embodiments, a spacer domain may also refer to and be synonymous with a hinge domain. The spacer domain is located between the binding domain and the transmembrane domain (TM). The spacer domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The spacer domain can be modified by replacing one or more cysteine ​​and / or proline residues in the naturally occurring immunoglobulin hinge domain with one or more other amino acid residues (e.g., one or more serine residues).

[0137] The "transmembrane domain" or "TM domain" refers to the hydrophobic portion of a chimeric antigen receptor polypeptide that anchors the polypeptide to the cell membrane. TM domains can originate from natural, synthetic, semi-synthetic, or recombinant sources.

[0138] "Intracellular signal transduction domain" refers to a polypeptide domain that participates in transducing information about the efficient binding of target antigens to chimeric antigen receptors expressed on immune effector cells into the immune effector cell to trigger one or more effector functions ("effector function" refers to a specific function of an immune effector cell), such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses triggered by antigen binding to receptors expressed on immune effector cells. "Intracellular signal transduction domain" includes polypeptide domains or functional fragments thereof that transduce effector function signals and direct the cell to perform specific functions. The term "intracellular signal transduction domain" refers to any truncated portion of an intracellular signal transduction domain that includes sufficient components to transduce effector function signals.

[0139] T cell activation can be thought to be mediated by two distinct intracellular signaling domains: the primary signaling domain, which initiates antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex); and the co-stimulatory signaling domain, which functions in an antigen-independent manner, providing secondary or co-stimulatory signals.

[0140] The "primary signal transduction domain" refers to the signal transduction domain that regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. A stimulatory primary signal transduction domain may contain one or more signal transduction motifs, known as immune receptor tyrosine activation motifs or ITAMs.

[0141] The term "co-stimulatory signal transduction domain" refers to the intracellular signal transduction domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, upon binding to antigens, provide a second signal required for the effective activation and function of T lymphocytes.

[0142] "Linker," "peptide linker," and "peptide linker" are used interchangeably, referring to multiple amino acid residues added between various polypeptide domains to achieve appropriate spacing, conformation, and function. Peptide linker sequences can be used to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds to form its appropriate secondary and tertiary structures, thereby enabling each polypeptide domain to perform its intended function. Linkers include "variable domain linking sequences," which are amino acid sequences that link two or more domains of an antibody or its antigen-binding fragment, providing a spacer function compatible with the interaction of two sub-binding domains, thus allowing the resulting polypeptide to maintain the same specific binding affinity to the same target molecule as antibodies containing the same light chain and / or heavy chain variable domains. The length of the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acids. Illustrative examples of linkers include, but are not limited to, the following amino acid sequences: TGEKP (SEQ ID NO: 2); (GGGGS) )n, where n = 1, 2, 3, 4 or 5 (SEQ ID NO: 3 and 976-979); EGKSSGSGSESKVD (SEQ ID NO: 4); KESGSSVSSEQLAQFRSLD (SEQ ID NO: 5); LRQRDGERP (SEQ ID NO: 6); LRQKDGGGSERP (SEQ ID NO: 7); LRQKD(GGGS)ERP (SEQ ID NO: 8), GEGTSTGSGGSGGSGGAD (SEQ ID NO: 9) and GSTGSSGKPGSGEGSTKG (SEQ ID NO: 10).

[0143] Other definitions are set forth throughout this disclosure.

[0144] C. Antibodies

[0145] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily 17 (TNFRSF17) and is highly expressed on plasma cells of patients with multiple myeloma (MM). Restricted expression of BCMA makes it a suitable therapeutic target for multiple myeloma. This disclosure contemplates antibodies that bind to BCMA and antigen-binding fragments thereof. An "antibody" is a polypeptide or antigen-binding fragment thereof comprising at least a light chain immunoglobulin variable region and / or a heavy chain immunoglobulin variable region, said polypeptide or antigen-binding fragment thereof specifically recognizing and binding one or more epitopes of a BCMA polypeptide, for example, SEQ ID NO: 1(MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR).

[0146] If the antibody or its antigen-binding fragment has a high affinity for the BCMA peptide or K a ≥ 10 5 M -1 If an antibody or its antigen-binding fragment binds to a BCMA peptide without significantly binding to other components present in the test sample, it is said to "specifically bind" to the BCMA peptide. Antibodies or their antigen-binding fragments can be classified as "high-affinity" or "low-affinity." "High-affinity" antibodies or their antigen-binding fragments refer to those that bind at least 10... 7 M -1 At least 10 8 M-1 At least 10 9 M -1 At least 10 10 M -1 At least 10 11 M -1 At least 10 12 M -1 Or at least 10 13 M -1 K a Antibodies that bind to BCMA. "Low-affinity" antibodies or their antigen-binding fragments refer to antibodies with an affinity of up to 10. 7 M -1 Up to 10 6 M -1 Up to 10 5 M -1 K a Antibodies that bind to BCMA. Alternatively, affinity can be defined as the equilibrium dissociation constant (K0) of a specific binding interaction. d ), the unit is M (for example, 10 -5 M to 10 -13 M).

[0147] Antibodies include polyclonal and monoclonal antibodies and their antigen-binding fragments; camel antibodies and human antibodies and their antigen-binding fragments; as well as chimeric antibodies, antibodies containing variable regions from non-human species and human constant regions, heteroconjugated antibodies and humanized antibodies, antibodies containing complementarity-determining regions (CDRs) from non-human species and human framework and constant regions and their antigen-binding fragments.

[0148] Chimeric antibodies, humanized antibodies, and human antibodies consist of two heavy chains and two light chains. Each heavy chain comprises one variable region (VH) and three constant regions (CH1, CH2, CH3), while each light chain comprises one variable region (VL) and one constant region (CL). Mammalian immunoglobulin heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Mammalian immunoglobulin light chains are classified as λ or κ.

[0149] The variable regions of light and heavy chains contain a “frame” region that is interrupted by three highly variable regions (also known as “complementary determinant regions” or “CDRs”).

[0150] The sequences of the framework regions of different light or heavy chains are relatively conserved within the same species (e.g., humans). Framework regions are used to locate and align CDRs in three-dimensional space for epitope binding. CDRs of each chain are numbered sequentially starting from the N-terminus and are often identified by the chain in which a particular CDR is located. Heavy chain CDRs are designated CDRH1, CDRH2, and CDRH3, while light chain CDRs are designated CDRL1, CDRL2, and CDRL3. Although the CDRs of different antibodies vary, the limited number of amino acid positions within a CDR that directly participate in antigen binding are called specificity-determining residues (SDRs).

[0151] CDRs can be defined or identified using conventional methods, such as based on Wu and Kabat. J Exp Med . 132(2):211-50 (1970) and Kabat and Wu, Ann New York Acad Sci The sequence 190:382–93 (1971) can be defined or identified, or according to Chothia and Lesk. J Mol. Biol . 196(4): 901-917 (1987) and Chothia et al., Nature. The structure of 342:877–83 (1989) is defined or identified. Padlan et al. FASEB J .9:133–9 (1995) and MacCallum et al., J Mol Biol 262:732–745 (1996) describes other definitions of the boundaries where the CDR overlaps with the Kabat CDR. Other methods for determining the CDR include the Gelfand numbering system, described in Gelfand and Kierkegaard. PNAS USA. 92:10884–8 (1995), Gelfand et al. Protein Eng 11:1015–25 (1998), and Gelfand et al., PNAS USA 93:3675–8 (1996); Honneger numbering system, described in Honegger and Plückthun, J Mol Biol 309:657–70 (2001); AbM numbering system, described in Abhinandan and Martin, Mol Immunol 45:3832–9 (2008); and the IMGT numbering system, described in Giudicelli et al., Nucleic Acids Res . 25:206–11 (1997), Lefranc, Immunol Today 18:509 (1997), and Lefranc et al., Dev Comp Immunol27:55–77 (2003). Proprietary and public procedures for identifying CDRs are available, such as abYsis (abysis.org / abysis / ) and IMGT / V-QUEST (imgt.org / IMGT_vquest).

[0152] “VL” or “V” L "VH" refers to the variable region of the immunoglobulin light chain or its antigen-binding fragment. H "" refers to the variable region of the immunoglobulin heavy chain or its antigen-binding fragment.

[0153] "Antigen-binding fragment" or "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. "Isolated antibody or its antigen-binding fragment" refers to an antibody or its antigen-binding fragment isolated from its natural environment, and / or an antibody or its antigen-binding fragment derived from natural, synthetic, semi-synthetic, or recombinant sources. Illustrative examples of antigen-binding fragments considered in the specific embodiments described herein include, but are not limited to: llama Ig, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv (bis-scFv), (scFv)2, microantibodies, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, disulfide-stabilized Fv proteins ("dsFv"), single-domain antibodies (sdAbs or nanobodies, such as camel VHH), other portions of full-length antibodies sufficient to achieve antigen binding, and combinations thereof.

[0154] "Heavy chain antibody" or "hcAb" refers to an antibody containing two heavy chain variable domains but no light chain. "Cameloid antibody" or "Cameloid Ig" refers to an hcAb isolated from camels, alpacas, or llamas, consisting of a homodimer composed of a heavy chain variable domain (VHH) and constant CH2 and CH3 domains. The terms "single-domain antibody," "sdAb," or "nanobody" used in this article refer to antibody fragments containing the smallest known antigen-binding unit of the heavy chain antibody variable region, such as cameloid VHH. "Humanized VHH" refers to a single-domain non-human VHH that has been humanized to reduce the potential immunogenicity of the antibody in human receptors.

[0155] A "single-chain Fv" or "scFv" antibody fragment contains the antibody's VH and VL domains, which are present in a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). Typically, scFv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows scFv to form the structure required for antigen binding.

[0156] In a specific implementation, the anti-BCMA antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH), which includes CDRH1, CDRH2, and CDRH3 of the antibody or its antigen-binding fragment as shown in Table 1; a peptide linker; and a light chain variable region (VL), which includes CDRL1, CDRL2, and CDRL3 of the antibody or its antigen-binding fragment as shown in Table 1.

[0157] In specific embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-linker-VH or VH-linker-VL),: a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 12, 13, and 14, a peptide linker, and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 16, 17, and 18; a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 22, 23, and 24, a peptide linker, and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 26, 27, and 28; a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 32, 33, and 34, a peptide linker, and a VL containing SEQ ID NO: 16, 17, and 18. VLs containing the amino acid sequences shown in SEQ ID NO: 36, 37, and 38 for CDRL1, CDRL2, and CDRL3; VHs containing the amino acid sequences shown in SEQ ID NO: 42, 43, and 44 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 46, 47, and 48 for CDRL1, CDRL2, and CDRL3; VHs containing the amino acid sequences shown in SEQ ID NO: 52, 53, and 54 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 56, 57, and 58 for CDRL1, CDRL2, and CDRL3; VHs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for CDRH2, CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for ... VLs containing CDRL1, CDRL2, and CDRL3 with amino acid sequences shown in SEQ ID NO: 66, 67, and 68; VHs containing CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 72, 73, and 74; VLs containing CDRL1, CDRL2, and CDRL3 with amino acid sequences shown in SEQ ID NO: 76, 77, and 78; VHs containing CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 82, 83, and 84; VLs containing CDRL1, CDRL2, and CDRL3 with amino acid sequences shown in SEQ ID NO: 86, 87, and 88;The peptide linker comprises a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 92, 93, and 94, and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 96, 97, and 98. In a specific embodiment, the peptide linker is selected from (GGGGS); n , where n = 1, 2, 3, 4 or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing at least 90% of the same amino acid sequence.

[0158] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 11; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 15; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0159] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 21; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 25; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0160] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 31; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 35; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0161] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 41; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 45; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0162] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 51; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 55; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0163] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 61; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 65; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0164] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 71; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 75; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0165] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 81; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 85; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0166] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): a VH comprising the amino acid sequence shown in SEQ ID NO: 91; a peptide connector; and a VL comprising the amino acid sequence shown in SEQ ID NO: 95; wherein the peptide connector is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0167] In a specific implementation, the anti-BCMA antibody or its antigen-binding fragment comprises an amino acid sequence represented by any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99 and 100, or an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with said sequence.

[0168] In a specific implementation, the anti-BCMA antibody or its antigen-binding fragment contains a VHH domain, which includes CDRH1, CDRH2 and CDRH3 of the antibodies or their antigen-binding fragments listed in Table 1.

[0169] In a specific embodiment, the anti-BCMA antibody or its antigen-binding fragment comprises: a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 102, 103, and 104; a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 106, 107, and 108; a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 110, 111, and 112; a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 114, 115, and 116; a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 118, 119, and 120; a VHH domain comprising SEQ ID NO: 104, 105, and 106; a VHH domain comprising SEQ ID NO: 106, 107, and 108; a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 104, 105, and 116; a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 118, 119, and 120; a VHH domain comprising SEQ ID NO: 104, 105, and 106; a VHH domain comprising SEQ ID NO: 106, 107, and 108; a VHH domain comprising SEQ ID NO: 104, 105, and 106; a VHH domain comprising SEQ ID NO: 104, 105, and 116; a VHH domain comprising The following structures contain VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 122, 123, and 124; VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 126, 127, and 128; VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 130, 131, and 132; VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 134, 135, and 136; VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 138, 139, and 140; and VHH domains of CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 142, 143, and 144.

[0170] In a specific embodiment, an anti-BCMA antibody or its antigen-binding fragment comprises VHH, wherein the VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141, or an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0171] Table 1

[0172] In specific embodiments, an anti-BCMA antibody comprising the amino acid sequence shown in Table 1 or an antigen-binding fragment thereof is conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from toxins, radioisotopes, RNA polymerase II inhibitors and / or RNA polymerase III inhibitors, and DNA damaging agents.

[0173] In a specific implementation, an anti-BCMA antibody comprising the amino acid sequence shown in Table 1, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent comprising a toxin. Illustrative examples of toxins considered in the specific implementation include, but are not limited to, saponins, diphtheria toxin, Pseudomonas exotoxin A, ricin A chain derivatives, small molecule toxins, and combinations thereof.

[0174] In a specific embodiment, an anti-BCMA antibody comprising the amino acid sequence shown in Table 1, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent comprising a radioactive isotope. Illustrative examples of radioactive isotopes considered in the specific embodiment include, but are not limited to, those mentioned above. 131 I, 90 Y、 177 Lu、 188 Re、 67 Cu、 213 Bi、 211 At and 227 Ac.

[0175] In specific embodiments, an anti-BCMA antibody comprising the amino acid sequence shown in Table 1 or an antigen-binding fragment thereof is conjugated to a cytotoxic agent comprising an RNA polymerase II and / or III inhibitor. Illustrative examples of RNA polymerase II and / or III inhibitors considered in specific embodiments include, but are not limited to, amatoxins, including but not limited to α-amatoxins, β-amatoxins, γ-amatoxins, ε-amatoxins, amatoxins, amatoxin amides, amatoxin nontoxic cyclic peptides, amatoxin acid, and any functional fragments, derivatives, or analogs thereof.

[0176] In a specific implementation, an anti-BCMA antibody comprising the amino acid sequence shown in Table 1, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent comprising a DNA-damaging agent. Illustrative examples of DNA-damaging agents considered in the specific implementation include, but are not limited to, anti-tubulin agents, DNA cross-linking agents, DNA alkylating agents, and mitotic disruptors.

[0177] D. Chimeric antigen receptor

[0178] A chimeric antigen receptor (CAR) is a fusion polypeptide that redirects the specificity of immune effector cells to the specificity of an antibody for a desired antigen (e.g., BCMA), thereby triggering proliferation, cytokine production, phagocytosis, or the production of molecules that mediate cell death that mediates the expression of the target antigen in a manner independent of major histocompatibility (MHC). As used herein, the term "chimeric" refers to a molecule composed of polypeptides or polynucleotides from two or more different sources.

[0179] This disclosure contemplates improved anti-BCMA CARs suitable for in vivo modification or ex vivo fabrication of immune effector cells to redirect cytotoxicity to BCMA-expressing cells (e.g., B cells). In various embodiments, the CAR includes a binding domain comprising one or more antibodies or antigen-binding fragments thereof that bind BCMA; a spacer domain; a transmembrane domain; and one or more intracellular signaling domains. In specific embodiments, the CAR includes an antibody or antigen-binding fragment thereof that binds BCMA, a spacer domain, a transmembrane domain, and a major signaling domain. In specific embodiments, the CAR includes an antibody or antigen-binding fragment thereof that binds BCMA, a spacer domain, a transmembrane domain, one or more co-stimulatory signaling domains, and a major signaling domain.

[0180] Illustrative examples of chimeric antigen receptor polypeptides are listed in SEQ ID NO: 165-860, and illustrative examples of polynucleotides encoding chimeric antigen receptor polypeptides are listed in SEQ ID NO: 905-944.

[0181] 1. Combining structural domains

[0182] In a specific implementation, the CAR includes an extracellular antigen-binding domain containing an antibody or antigen-binding fragment thereof that specifically binds to a human BCMA polypeptide. The terms "binding domain" and "extracellular antigen-binding domain" are used interchangeably to refer to one or more antibodies or antigen-binding fragments thereof that bind BCMA. The binding domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0183] In a specific implementation, the CAR includes a binding domain that contains one or more single-stranded variable fragments (scFvs) and / or VHH domains that bind BCMA. In a specific implementation, the CAR includes a binding domain that contains one or more scFvs that bind BCMA. In a specific implementation, the CAR includes a binding domain that contains one or more VHH domains that bind BCMA.

[0184] In a specific implementation, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment thereof, the anti-BCMA antibody or its antigen-binding fragment comprising a heavy chain variable region (VH), the heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 of the antibody or its antigen-binding fragment as shown in Table 1; a peptide linker; and a light chain variable region (VL), the light chain variable region comprising CDRL1, CDRL2 and CDRL3 of the antibody or its antigen-binding fragment as shown in Table 1; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain.

[0185] In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 12, 13, and 14; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 16, 17, and 18; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 22, 23, and 24; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 26, 27, and 28; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 32, 33, and 34; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 36, 37, and 38; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 42, 43, and 44; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 46, 47, and 48; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain.In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 52, 53, and 54; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 56, 57, and 58; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a preferred embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 62, 63, and 64; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 66, 67, and 68; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 72, 73, and 74; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 76, 77, and 78; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), the anti-BCMA antibody or its antigen-binding fragment comprising a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 82, 83, and 84; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 86, 87, and 88; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain.In a specific embodiment, the CAR comprises an anti-BCMA antibody or its antigen-binding fragment in any orientation (e.g., VL-connector-VH or VH-connector-VL), wherein the anti-BCMA antibody or its antigen-binding fragment comprises a VH containing CDRH1, CDRH2, and CDRH3 having the amino acid sequences shown in SEQ ID NO: 92, 93, and 94; a peptide connector; and a VL containing CDRL1, CDRL2, and CDRL3 having the amino acid sequences shown in SEQ ID NO: 96, 97, and 98; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the peptide connector is selected from (GGGGS). n , where n = 1, 2, 3, 4 or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing at least 90% of the same amino acid sequence.

[0186] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 11; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; VL containing the amino acid sequence shown in SEQ ID NO: 15; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0187] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or its antigen-binding fragment thereof, said anti-BCMA antibody or its antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 21; and a polypeptide adapter selected from the following: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 25; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0188] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or its antigen-binding fragment thereof, said anti-BCMA antibody or its antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 31; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 35; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0189] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 41; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 45; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0190] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 51; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 55; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0191] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 61; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 65; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0192] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or its antigen-binding fragment thereof, said anti-BCMA antibody or its antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 71; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 75; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0193] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 81; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their amino acid sequence variants containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 85; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0194] In a specific embodiment, the CAR comprises, in any orientation (e.g., VL-connector-VH or VH-connector-VL): an anti-BCMA antibody or its antigen-binding fragment thereof, said anti-BCMA antibody or its antigen-binding fragment comprising a VH containing the amino acid sequence shown in SEQ ID NO: 91; and a polypeptide adapter selected from the group consisting of (GGGGS). n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and variants thereof containing 95% identical amino acid sequences; and VL containing the amino acid sequence shown in SEQ ID NO: 95; spacer domain; transmembrane domain; co-stimulatory signal transduction domain; and major signal transduction domain.

[0195] In a specific implementation, the CAR comprises an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising an amino acid sequence shown in any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99 and 100 or an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain.

[0196] In specific embodiments, the CAR comprises one or more BCMA-binding VHH domains, wherein the VHH domains comprise CDRH1, CDRH2, and CDRH3 of the antibody or its antigen-binding fragment as shown in Table 1; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In specific embodiments, the CAR comprises a VHH domain, wherein the VHH domains comprise CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 102, 103, and 104; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In specific embodiments, the CAR comprises a VHH domain, wherein the VHH domains comprise CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 106, 107, and 108; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 110, 111, and 112; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 114, 115, and 116; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 118, 119, and 120; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 122, 123, and 124; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 126, 127, and 128; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain.In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 130, 131, and 132; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 134, 135, and 136; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 138, 139, and 140; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes a VHH domain comprising CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 142, 143, and 144; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain. In a specific embodiment, the CAR includes one or more VHH domains shown in SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141, or amino acid sequences having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with them; a spacer domain; a transmembrane domain; a co-stimulatory signal transduction domain; and a major signal transduction domain.

[0197] 2. Spacing structure domain

[0198] The chimeric antigen receptor considered in this paper includes a spacer domain. This spacer domain is positioned between the extracellular antigen-binding domain and the transmembrane domain of the CAR. The spacer domain functions to position the extracellular antigen-binding domain away from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation. The spacer domain may be derived from the hinge or stalk domain of a natural polypeptide, or from synthetic, semi-synthetic, or recombinant sources.

[0199] In a specific embodiment, the CAR includes a spacer domain comprising a hinge and / or stalk domain, the hinge and / or stalk domain being isolated from, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, CD4, CD7, CD8α, CD8β, CD28, CD134, CD137, CD152, and CD278. In a specific embodiment, the CAR includes a spacer domain comprising a naturally occurring immunoglobulin hinge region isolated from IgG1, IgG2, IgG3, or IgG4, optionally combined with one or more heavy chain constant regions such as CH2 and / or CH3.

[0200] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, comprising the amino acid sequences shown in Table 1; a spacer domain selected from the CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, and IgG4 hinge; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0201] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, which comprises the amino acid sequences shown in Table 1; a spacer domain, which comprises the amino acid sequences shown in Table 2; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0202] Table 2

[0203] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, which comprises the amino acid sequences shown in Table 1; a spacer domain, which comprises the amino acid sequence shown in any one of SEQ ID NO: 145, 146, 147, 148, 149 and 150; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0204] 3. Transmembrane domain

[0205] The chimeric antigen receptor considered in this paper includes a transmembrane domain. The transmembrane domain is a hydrophobic domain that fuses the extracellular and intracellular portions of the CAR and anchors the CAR to the plasma membrane of immune effector cells. The transmembrane domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. In specific embodiments, the CAR also includes a short oligopeptide or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, positioned between the transmembrane domain and the intracellular domain of the CAR.

[0206] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, comprising the amino acid sequence shown in Table 1; a spacer domain; a transmembrane domain isolated from or derived from a polypeptide selected from: α, β, γ, or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death protein 1 (PDCD1); one or more co-stimulatory signal transduction domains; and a major signal transduction domain.

[0207] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, which comprises the amino acid sequences shown in Table 1; a spacer domain; a transmembrane domain, which comprises the amino acid sequences shown in Table 3; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0208] Table 3

[0209] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, which comprises the amino acid sequences shown in Table 1; a spacer domain; a transmembrane domain, which comprises the amino acid sequences shown in any one of SEQ ID NO: 151, 152, 153, 154, 155, 156 and 157; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0210] 4. Intracellular signal transduction domains

[0211] The chimeric antigen receptors considered in this paper contain one or more intracellular signaling domains that function to transduce extracellular antigen recognition signals into immune effector cells and trigger one or more effector cell functions, including but not limited to activation, cytokine production, proliferation, and cytotoxic activity. T cell activation is mediated by two distinct classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex) and a co-stimulatory signaling domain that provides secondary or co-stimulatory signals in an antigen-independent manner. The intracellular primary and co-stimulatory signaling domains can be tandemly linked to the C-terminus of the transmembrane domain in any order.

[0212] In a specific implementation, the CAR includes one or more intracellular signal transduction domains, which include one or more co-stimulatory signal transduction domains and a primary signal transduction domain.

[0213] The major signal transduction domain regulates primary activation of the TCR complex in a stimulatory or inhibitory manner. The major signal transduction domain acting in a stimulatory manner may contain a signal transduction motif known as an immune receptor tyrosine activation motif or ITAM. Major signal transduction domains containing one or more ITAMs can be obtained, isolated, or derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequences shown in Table 1; a spacer domain; a transmembrane domain; a major signal transduction domain isolated from a polypeptide selected from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d; and optionally one or more co-stimulatory signal transduction domains.

[0214] Costimulatory signaling domains provide the second signal required for the effective activation and function of immune effector cells after binding to antigens. Costimulatory signaling domains can be obtained, isolated, or derived from a selection of costimulatory molecules, including: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

[0215] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, comprising the amino acid sequence shown in Table 1; a spacer domain; a transmembrane domain; a major signal transduction domain isolated from a polypeptide selected from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d; and optionally one or more co-stimulatory signal transduction domains isolated from a co-stimulatory molecule selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134(OX40), CD137(4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25 and ZAP70.

[0216] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, which comprises the amino acid sequences shown in Table 1; a spacer domain; a transmembrane domain; a major signal transduction domain; and one or more co-stimulatory signal transduction domains, which comprise the amino acid sequences shown in Table 4.

[0217] Table 4

[0218] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising the amino acid sequences shown in Table 1; a spacer domain; a transmembrane domain; one or more co-stimulatory domains comprising the amino acid sequence shown in any one of SEQ ID NO: 159, 160, 161, 162, 163 and 164 or an amino acid sequence that is at least 95% identical thereto; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical thereto.

[0219] E. Explanatory chimeric antigen receptor

[0220] In a specific implementation, the CAR comprises: an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising the amino acid sequence listed in Table 1; a spacer domain comprising a hinge domain or fragment thereof selected from the following: CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, and IgG4 hinge; and a transmembrane domain isolated from or derived from a polypeptide selected from: α, β, γ, or δ chains of a T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α. CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, AMN, and PDCD1; one or more co-stimulatory signal transduction domains isolated from the following co-stimulatory molecules: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25 and ZAP70; and major signal transduction domains isolated from CD3ζ, CD22, CD79a, CD79b or CD66d.

[0221] In a specific implementation, the CAR comprises an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising the amino acid sequence shown in Table 1; a spacer domain comprising a hinge domain or a fragment thereof selected from the CD8α hinge, CD28 hinge, IgG1 hinge and IgG4 hinge; a CD8α or CD28 transmembrane domain; a CD134, CD137 or CD278 co-stimulatory domain; and a CD3ζ primary signal transduction domain.

[0222] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, and 188. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212.

[0223]

[0224] In a specific embodiment, the CAR comprises: an antibody or antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 30; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, and 236. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, and 260.

[0225]

[0226] In a preferred embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 39; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a preferred embodiment, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283 and 284.

[0227]

[0228] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 40; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, and 308.

[0229]

[0230] In a specific embodiment, the CAR comprises: an antibody or antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, and 332. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, and 356.

[0231]

[0232] In a preferred embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 59; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a preferred embodiment, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379 and 380.

[0233]

[0234] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 60; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, and 404.

[0235]

[0236] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 69; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427 and 428.

[0237]

[0238] In a preferred embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 70; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a preferred embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, and 452. In a more preferred embodiment, the CAR comprises the amino acid sequence listed in SEQ ID NO: 429, 432, 435, or 438.

[0239]

[0240] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 79 or SEQ ID NO: 80; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, and 476. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, and 500.

[0241]

[0242] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 89; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, and 524.

[0243]

[0244] In a preferred embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 90; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a preferred embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, and 548.

[0245]

[0246] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 99 or SEQ ID NO: 100; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, and 572. In a specific embodiment, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, and 596.

[0247]

[0248] In a preferred embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 101; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a preferred embodiment, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, and 620.

[0249]

[0250] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 105; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, and 644.

[0251]

[0252] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 109; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, and 668.

[0253]

[0254] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 113; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, and 692.

[0255]

[0256] In a preferred embodiment, the CAR comprises: an antibody or antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 117; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a preferred embodiment, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, and 716.

[0257]

[0258] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 121; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, and 740.

[0259]

[0260] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 125; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, and 764.

[0261]

[0262] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 129; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, and 788.

[0263]

[0264] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 133; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, and 812.

[0265]

[0266] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 137; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, and 836.

[0267]

[0268] In a specific embodiment, the CAR comprises: an antibody or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 141; a spacer domain comprising the amino acid sequence shown in SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148 or SEQ ID NO: 150; a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 151 or SEQ ID NO: 153; a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 162; and a major signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 158. In a specific implementation, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, and 860.

[0269]

[0270] F. Polypeptide

[0271] In the specific implementations, peptides, fusion peptides, and peptide variants are considered. Exemplary peptides considered herein include, but are not limited to, antibodies and their antigen-binding fragments, fusion peptides, bispecific antibodies, bispecific T-cell conjugates (BiTEs), antibody conjugates, chimeric antigen receptors (CARs) and their components, as well as variants and / or fragments thereof, such as SEQ ID NO: 1-873 and 954-979. Peptides considered herein also include peptides encoded by the polynucleotide sequences shown in any one of SEQ ID NO: 874-953.

[0272] Unless otherwise specified, the terms “polypeptide,” “peptide,” “protein,” and “peptide” are used interchangeably and, by convention, refer to an amino acid sequence. In specific embodiments, “polypeptide” refers to a fusion polypeptide or a polypeptide variant. Polypeptides can be prepared using any of a variety of known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length; for example, they can comprise a full-length protein sequence, a full-length protein fragment, or a fusion protein, and can include post-translational modifications such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, including both naturally occurring and non-natural modifications.

[0273] As used in this article, "isolated peptide," "isolated protein," or "isolated polypeptide" refers to polypeptide molecules that are isolated, separated, and / or purified from the cellular environment and from their binding with other cellular components, i.e., they do not bind significantly to substances in vivo.

[0274] Peptides include “peptide variants.” In specific embodiments, peptide variants are referred to as “modified peptides.” Peptide variants may differ from naturally occurring peptides in terms of substitutions, deletions, additions, and / or insertions of one or more amino acids. For example, in specific embodiments, it may be necessary to modulate one or more biological activities of a chimeric antigen receptor by introducing one or more amino acid substitutions, deletions, additions, and / or insertions into the peptide. Such variants are naturally occurring or synthetically produced. In specific embodiments, peptides include peptide variants that have at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity with any reference sequence considered herein, typically wherein the variant retains at least one biological activity of the reference sequence.

[0275] Peptide variants include “peptide fragments.” Illustrative examples of peptide fragments include, but are not limited to, binding domains, hinges, transmembrane domains, intracellular domains, etc. In a particular embodiment, the peptide fragment is a biologically active peptide fragment. As used herein, the term “biologically active peptide fragment” refers to a peptide fragment that retains at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% of the activity of a naturally occurring peptide. In some embodiments, the peptide fragment comprises an amino acid sequence of at least 5 to about 500 amino acids in length. It should be understood that, in some embodiments, the fragment length is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, or 500 or more amino acids. In specific embodiments, the polypeptide fragment comprises an antibody or antigen-binding fragment of BCMA that binds to it.

[0276] In specific embodiments, the polypeptide comprises one or more amino acid substitutions, deletions, truncations, or insertions, which are performed using methods known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA. 82:488-492. (1985)), Kunkel et al., (Methods in Enzymol, 154: 367-382. (1987)), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, 4th Edition, Benjamin / Cummings, Menlo Park, Calif. (1987)) and incorporated herein by reference. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC (1978)).

[0277] In some embodiments, the peptide variant contains one or more conserved substitutions. A “conserved substitution” refers to the replacement of one amino acid with another amino acid of similar properties. Guidelines for determining which amino acid residues can be replaced, inserted, or deleted can be found using computer programs well known in the art (e.g., DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software). In specific embodiments, the amino acid changes in the peptide variants considered herein include one or more conserved amino acid substitutions. Conserved amino acid substitutions include replacing an amino acid with an amino acid having a related side chain. Naturally occurring amino acids are generally classified into four classes: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively referred to as aromatic amino acids. In specific embodiments, a conserved amino acid substitution refers to the replacement of an amino acid within the same group or family. Those skilled in the art will recognize that, generally, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0278] In specific implementations, conserved amino acid substitution refers to the substitution of an amino acid with a similar hydrophilicity / hydrophobicity index or score. The importance of the hydrophilicity amino acid index in conferring biological functions of protein-protein interactions is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid is assigned a hydrophilicity / hydrophobicity index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values ​​are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​disulfide (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In a specific embodiment, conserved amino acid substitution refers to the substitution of amino acids with similar hydrophilicity or hydrophobicity indices or scores. In specific embodiments, it is preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±2, particularly preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±1, and even more preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±0.5. It is also understood in the art that, based on hydrophilicity, the same amino acids can be effectively replaced.

[0279] In a specific implementation, conserved amino acid substitution refers to the substitution of amino acids with similar hydrophilicity indices or scores. According to the detailed specifications of U.S. Patent No. 4,554,101, the hydrophilicity values ​​of the amino acid residues are as follows: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In specific embodiments, conservative amino acid substitution refers to replacing amino acids with similar hydrophilicity indices or scores. In specific embodiments, substitution of amino acids with a hydrophilicity index within ±2 is preferred, within ±1 is particularly preferred, and within ±0.5 is even more particularly preferred.

[0280] In specific implementations, conserved amino acid substitutions can be based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc.

[0281] In a specific embodiment, the polypeptide comprises: an anti-BCMA antibody or an antigen-binding fragment thereof, the anti-BCMA antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), the heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 of the antibody or antigen-binding fragment thereof as shown in Table 1; a polypeptide linker; and a light chain variable region (VL), the light chain variable region comprising CDRL1, CDRL2 and CDRL3 of the antibody or antigen-binding fragment thereof as shown in Table 1; and optionally a polypeptide linker and an anti-CD3 antibody.

[0282] In specific embodiments, a polypeptide comprising an anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-linker-VH or VH-linker-VL),: a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 12, 13, and 14, a polypeptide linker, and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 16, 17, and 18; a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 22, 23, and 24, a polypeptide linker, and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 26, 27, and 28; a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 32, 33, and 34, a polypeptide linker, and a VL containing SEQ ID NO: 16, 17, and 18, a polypeptide linker; a VL containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 16, 17, and 18, a polypeptide linker; a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 16, 17, and 18, a polypeptide linker; a VL ... VLs containing the amino acid sequences shown in SEQ ID NO: 36, 37, and 38 for CDRL1, CDRL2, and CDRL3; VHs containing the amino acid sequences shown in SEQ ID NO: 42, 43, and 44 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 46, 47, and 48 for CDRL1, CDRL2, and CDRL3; VHs containing the amino acid sequences shown in SEQ ID NO: 52, 53, and 54 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 56, 57, and 58 for CDRL1, CDRL2, and CDRL3; VHs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for CDRH1, CDRH2, and CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for CDRH2, CDRH3, and VLs containing the amino acid sequences shown in SEQ ID NO: 62, 63, and 64 for ... VLs containing CDRL1, CDRL2, and CDRL3 with amino acid sequences shown in SEQ ID NO: 66, 67, and 68; VHs containing CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 72, 73, and 74; VLs containing CDRL1, CDRL2, and CDRL3 with amino acid sequences shown in SEQ ID NO: 76, 77, and 78; VHs containing CDRH1, CDRH2, and CDRH3 with amino acid sequences shown in SEQ ID NO: 82, 83, and 84; VLs containing CDRL1, CDRL2, and CDRL3 with amino acid sequences shown in SEQ ID NO: 86, 87, and 88;The formulation comprises a VH containing CDRH1, CDRH2, and CDRH3 with the amino acid sequences shown in SEQ ID NO: 92, 93, and 94; a peptide linker; and a VL containing CDRL1, CDRL2, and CDRL3 with the amino acid sequences shown in SEQ ID NO: 96, 97, and 98; and optionally, a formulation comprising a peptide linker and an anti-CD3 antibody. In a specific embodiment, the peptide linker is selected from: (GGGGS); n , where n = 1, 2, 3, 4 or 5; GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing at least 90% of the same amino acid sequence.

[0283] In a specific embodiment, the polypeptide comprising an anti-BCMA antibody or its antigen-binding fragment comprises, in any orientation (e.g., VL-linker-VH or VH-linker-VL),: a VH comprising the amino acid sequence shown in any one of SEQ ID NO: 11, 21, 31, 41, 51, 61, 71, 81, and 91; a polypeptide linker; and a corresponding VL comprising the amino acid sequence shown in SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, and 95; and optionally, a polypeptide linker and an anti-CD3 antibody; wherein the polypeptide linker is selected from: (GGGGS) n , where n = 1, 2, 3, 4 or 5, GETSTGSGGSGGSGGAD, GSTGSGKPGSGEGSTKG and their variants containing 95% identical amino acid sequences.

[0284] In a specific embodiment, the polypeptide comprising an anti-BCMA antibody or an antigen-binding fragment thereof comprises an amino acid sequence shown in any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99 and 100, or an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; and optionally a polypeptide linker and an anti-CD3 antibody.

[0285] In a specific embodiment, the polypeptide comprises an anti-BCMA antibody or an antigen-binding fragment thereof, the anti-BCMA antibody or antigen-binding fragment thereof comprising a VHH domain comprising CDRH1, CDRH2 and CDRH3 of the antibodies or antigen-binding fragments thereof listed in Table 1; and optionally a polypeptide linker and an anti-CD3 antibody.

[0286] In a specific embodiment, the polypeptide comprises an anti-BCMA antibody or its antigen-binding fragment, wherein the anti-BCMA antibody or its antigen-binding fragment comprises: a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 102, 103, and 104 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 106, 107, and 108 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 110, 111, and 112 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 114, 115, and 116 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 118, 119, and 120 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 106, 107, and 108 of CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 106, 107, and 108 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 114, 115, and 116 of CDRH1, CDRH2, and CDRH3; a VHH domain comprising the amino acid sequences shown in SEQ ID NO: 118, 119, and 120 ... The VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 122, 123, and 124; the VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 126, 127, and 128; the VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 130, 131, and 132; the VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 134, 135, and 136; the VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 138, 139, and 140; the VHH domains of CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 142, 143, and 144; and optionally, a peptide linker and an anti-CD3 antibody.

[0287] In a specific embodiment, the polypeptide comprises an anti-BCMA antibody or its antigen-binding fragment thereof, said anti-BCMA antibody or its antigen-binding fragment comprising VHH, said VHH comprising an amino acid sequence shown in any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141 or an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; and optionally a polypeptide linker and an anti-CD3 antibody.

[0288] The polypeptides considered in specific embodiments include fusion polypeptides. In specific embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides may contain one or more polypeptide domains or fragments, including but not limited to signal peptides, antibodies or antigen-binding fragments thereof, polypeptide linkers, spacer domains, transmembrane domains, intracellular signal transduction domains, and polypeptide cleavage signals. Fusion proteins and polypeptides are typically C-terminus-N-terminus linked together, although they can also be C-terminus-C-terminus, N-terminus-N-terminus, or N-terminus-C-terminus linked together. Fusion polypeptides and fusion proteins refer to polypeptides containing at least two, three, four, five, six, seven, eight, nine, or ten polypeptide fragments.

[0289] In a specific embodiment, the fusion polypeptide, such as CAR, comprises a signal peptide as shown in any of SEQ ID NO: 861-873, which is subsequently cleaved from the fusion polypeptide. The signal peptide is a short N-terminal sequence of 16 to 30 amino acids in length from a newly synthesized polypeptide chain that mediates protein targeting of the endoplasmic reticulum (ER) membrane. Typically, the signal peptide is cleaved by a signal peptidase (a heterooligomeric polypeptide complex) during translation. In a specific embodiment, the polypeptide comprises a signal peptide. In a preferred embodiment, the polynucleotide encoding the polypeptide comprises a polynucleotide encoding the signal peptide; and the translated polypeptide does not contain a signal peptide. Table 5 lists exemplary signal peptides.

[0290] Table 5: Exemplary signal peptides

[0291] In a specific embodiment, the polypeptide comprises a signal peptide represented by any one of SEQ ID NO: 861-973 and a chimeric antigen receptor comprising an amino acid sequence represented by any one of SEQ ID NO: 165-860.

[0292] In a specific embodiment, the polypeptide comprises a signal peptide shown in any one of SEQ ID NO: 861-973 and a chimeric antigen receptor encoded by a polynucleotide sequence shown in any one of SEQ ID NO: 904-944.

[0293] The fusion polypeptide may optionally include a polypeptide linker as described elsewhere herein, which can be used to link one or more polypeptides or domains within the polypeptide. Exemplary linkers are listed in SEQ ID NO: 2-10.

[0294] In a specific implementation, two or more polypeptides may be represented as a fusion polypeptide, which includes one or more polypeptide cleavage signals disposed between the two or more polypeptides.

[0295] Exemplary peptide cleavage signals include, but are not limited to, protease cleavage sites, nuclease cleavage sites, and ribosome-jumping peptides or self-cleaving viral peptides (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; deFelipe and Ryan, 2004. Traffic, 5(8); 616-26; and Scymczak et al., (2004) Nature Biotech. 5, 589-594).

[0296] Exemplary protease cleavage sites include, but are not limited to: potato virus Y (NIa) protease (e.g., tobacco erosion virus protease), potato virus Y (HC) protease, potato virus Y (PI) (P35) protease, byovirus NIa protease, byovirus RNA-2 encoded protease, foot-and-mouth disease virus (FMD) L protease, enterovirus 2A protease, rhinovirus 2A protease, small RNA virus (PRV) 3C protease, cowpea mosaic virus (CVV) 24K protease, nematode virus (NNV) 24K protease, RTSV (rice tungal virus) 3C-like protease, PYVF (European windbreak virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase cleavage sites.

[0297] Illustrative examples of ribosomal jumping peptides include, but are not limited to, viral 2A peptides or sequences (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In a specific embodiment, the viral 2A peptide is a foot-and-mouth disease virus 2A peptide, a potato virus 2A peptide, or a myocarditis virus 2A peptide.

[0298] In one embodiment, the viral 2A peptide is selected from: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, thosea asigna virus (TaV) 2A peptide, porcine parvovirus-1 (PTV-1) 2A peptide, Theylvirus 2A peptide, and encephalomyelitis virus 2A peptide.

[0299] Illustrative examples of viral 2A sequences include, but are not limited to: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 956); ATNFSLLKQAGDVEENPGP (SEQ ID NO: 957); LLKQAGDVEENPGP (SEQ ID NO: 958); GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 959); EGRGSLLTCGDVEENPGP (SEQ ID NO: 960); LLTCGDVEENPGP (SEQ ID NO: 961); GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 962); QCTNYALLKLAGDVESNPGP (SEQ ID NO: 963); LLKLAGDVESNPGP (SEQ ID NO: 964); GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 956); EGRGSLLTCGDVEENPGP (SEQ ID NO: 957); LLKQAGDVEENPGP (SEQ ID NO: 958); GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 959); EGRGSLLTCGDVEENPGP (SEQ ID NO: 960); LLTCGDVEENPGP (SEQ ID NO: 961); GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 962); QCTNYALLKLAGDVESNPGP (SEQ ID NO: 963); LLKLAGDVESNPGP (SEQ ID NO: 964); GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 965). 965); VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 966); LLNFDLLKLAGDVESNPGP (SEQ ID NO: 967); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 968); NFDLLKLAGDVESNPGP (SEQ ID NO: 969); QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 970); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 971); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 972); LNFDLLKLAGDVESNPGP (SEQ ID NO: 973); (SEQ ID NO: 974); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 975).

[0300] G. Polynucleotides

[0301] Various implementation schemes have been considered to include or encode antibodies and their antigen-binding fragments, bispecific antibodies, BiTEs, antibody conjugates, chimeric antigen receptors, vectors, promoters, enhancers, Kozak sequences, polyadenylation signals, untranslated regions, and post-transcriptional response elements, as well as other polynucleotides.

[0302] As used herein, the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded, and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-mRNA, mRNA, RNA, circRNA, synthetic RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, gRNA, viral gRNA, positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), doggybone DNA (dbDNA), linear DNA, circular DNA, PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymeric form of at least 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 5000, 10000, or 15000 or more nucleotides, which are ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide, including all intermediate lengths. It is readily understood that in this document, "intermediate length" refers to any length between reference values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0303] As used herein, "isolated polynucleotide" refers to a polynucleotide isolated or purified from naturally occurring flanking sequences. In specific embodiments, the isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, a polynucleotide obtained or derived from a recombinant source, or other polynucleotides that do not exist in nature or are artificially manufactured.

[0304] In specific implementations, the polynucleotides considered herein are polynucleotide variants. As used herein, the terms "polynucleotide variant" and "variant," etc., refer to a polynucleotide that has substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also include polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of one or more nucleotides. Therefore, the terms "polynucleotide variant" and "variant" encompass polynucleotides with the addition or deletion of one or more nucleotides, or modified or substituted with different nucleotides. In this regard, it is well understood in the art that certain alterations can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or that the function or activity of the altered polynucleotide is modulated. In a specific implementation, the polynucleotide or polynucleotide variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence.

[0305] In specific implementations, polynucleotide variants include polynucleotide fragments encoding biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a fragment with a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250. A polynucleotide fragment of 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides, said polynucleotide fragment encoding a polypeptide variant that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide. A polynucleotide fragment refers to a polynucleotide encoding a polypeptide having an N-terminal deletion, a C-terminal deletion, and / or an internal deletion or substitution of one or more amino acids in a naturally occurring or recombinant polypeptide.

[0306] As used herein, the phrase “sequence identity” or, for example, “sequence with 50% identity”, refers to the degree to which sequences are identical on a nucleotide-to-nucleotide or amino acid-to-amino acid basis within a comparison window. The “sequence identity percentage” can be calculated by comparing two best-aligned sequences within a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to produce the number of matching positions. The number of matching positions is then divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to obtain the sequence identity percentage. In specific implementations, the polynucleotides and polypeptides have at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity with any of the reference sequences described herein, such as SEQ ID NO: 1-979.

[0307] Illustrative examples of polynucleotides include, but are not limited to, the polynucleotide sequences shown in any one of SEQ ID NO: 874-953, and the polynucleotides encoding the polypeptides shown in SEQ ID NO: 1-873 and 954-979.

[0308] In various embodiments, the polynucleotide encodes a polypeptide comprising any one of the amino acid sequences shown in SEQ ID NO: 1-873 and 954-979.

[0309] In various embodiments, the polynucleotide encodes an antigen or an antigen-binding fragment thereof comprising any one of the amino acid sequences shown in SEQ ID NO: 11-144. In a specific embodiment, the polynucleotide encoding the antigen or an antigen-binding fragment thereof comprises any one of the polynucleotide sequences shown in SEQ ID NO: 874-893.

[0310] Table 6 lists the SEQ ID NO. and associated nucleic acid sequence encoding anti-BCMA antibody or its antigen-binding fragment, as well as the corresponding amino acid SEQ ID NO. (AA SEQ ID NO.) encoded by the nucleic acid sequence.

[0311] Table 6

[0312] In various embodiments, the polynucleotide encodes a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising any one of the amino acid sequences shown in SEQ ID NO: 11-144. In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof, said anti-BCMA antibody or antigen-binding fragment comprising any one of the amino acid sequences shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141; and optionally a polypeptide linker and an anti-CD3 antibody.

[0313] In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 165-860. In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837. In a specific embodiment, the polynucleotide encoding the chimeric antigen receptor comprises the polynucleotide sequence shown in any one of SEQ ID NO: 905-944.

[0314] In specific implementations, the polynucleotide encoding the chimeric antigen receptor may be codon-optimized. As used herein, the term "codon optimization" refers to replacing codons in a polynucleotide encoding a polypeptide to modulate the polypeptide's expression, stability, and / or activity. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) differences in codon preference between two or more organisms or genes, or based on artificially constructed bias tables; (ii) differences in the degree of codon preference within a specific organism, gene, or genome; (iii) systematic variations of codons (including their context); (iv) codon differences based on their corresponding decoded tRNAs; (v) codon differences based on GC content (whether overall or at specific positions within triplet codons); (vi) differences in similarity to reference sequences (e.g., naturally occurring sequences); (vii) differences in codon frequency cutoff values; (viii) the structural characteristics of the mRNA transcribed from the DNA sequence; (ix) prior knowledge about the function of the DNA sequence (the construction of codon substitution sets is based on such knowledge); (x) systematic variations for each amino acid codon subset; and / or (xi) individual removal of unexpected translation initiation sites.

[0315] A “nucleic acid cassette,” “expression cassette,” or “nucleic acid expression cassette” refers to a polynucleotide sequence sufficient to transcribe RNA, which is ultimately translated into a polypeptide. In specific embodiments, the nucleic acid cassette contains a target polynucleotide, i.e., a polynucleotide encoding a polypeptide, such as a CAR. Nucleic acid expression cassettes considered in specific embodiments contain one or more expression control sequences, such as promoters, enhancers, polyadenylated sequences, and one or more target polynucleotides. In specific embodiments, the vectors envisioned herein include one or more nucleic acid cassettes. In specific embodiments, the nucleic acid cassette is directed into the vector to achieve transcription of the target polynucleotide.

[0316] In specific embodiments, the polynucleotide encoding the polypeptide may be combined with other polynucleotide sequences, such as expression control sequences, promoters and / or enhancers, untranslated regions (UTRs), polynucleotides encoding signal peptides, Kozak sequences, polyadenylation signals, restriction endonuclease sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites, stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides or epitope tags, as disclosed elsewhere herein or known in the art.

[0317] Polynucleotides can be prepared, manipulated, expressed, and / or delivered using any of a variety of well-established techniques known and available in the art. To express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector.

[0318] In specific implementations, polynucleotides are inserted into nonviral vectors. Illustrative examples of nonviral vectors include, but are not limited to, autonomously replicating sequences; plasmids; phage particles; granules; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages, such as λ phage or M13 phage; and transposon elements, including but not limited to piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives.

[0319] In a specific implementation, a polynucleotide is inserted into the viral vector. Illustrative examples of viral vectors include, but are not limited to: adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, rhabdovirus (e.g., rabies virus, vesicular virus) vectors, paramyxovirus (e.g., hennipa virus, measles virus, respiratory virus, rubella virus) vectors, herpes simplex virus (e.g., HSV-1, HSV-2) vectors, vaccinia virus vectors, and retroviral vectors, preferably lentiviral vectors (LVV).

[0320] In a specific implementation, the vector includes a polynucleotide that contains or encodes one or more exogenous, endogenous, or heterologous expression control sequences, the polynucleotide being operatively linked to a polynucleotide encoding one or more polynucleotides and / or polypeptides as considered herein.

[0321] The “expression control sequence,” “control element,” or “regulatory sequence” considered in the specific implementation plan includes, but is not limited to, promoters, enhancers, translation initiation signals (Shine Dalgamo sequences or Kozak sequences), introns, polyadenylation signals, and 5ˊ and 3ˊ untranslated regions, all of which can interact with host cell proteins to perform transcription and translation.

[0322] As used herein, the term "promoter" refers to the recognition site of a polynucleotide (DNA or RNA) that RNA polymerase binds to. RNA polymerase initiates and transcribes polynucleotides operatively linked to a promoter. In specific embodiments, promoters functioning in mammalian cells include an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site and / or another sequence located 70 to 80 bases upstream of the transcription start site, namely the CNCAAT region, where N can be any nucleotide. The term "enhancer" refers to a segment of DNA containing a sequence capable of enhancing transcription, and in some cases, its function can be independent of its orientation relative to another control sequence. Enhancers can function synergistically or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA containing a sequence capable of simultaneously functioning as a promoter and enhancer.

[0323] The term "operably linked" refers to a juxtaposition where the components are in a relationship that allows them to function in a intended manner. In one embodiment, the term refers to a functional link between an expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence encoding a polypeptide, wherein the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0324] Illustrative universal expression control sequences applicable to specific implementation schemes include, but are not limited to, the β-actin promoter, the cytomegalovirus (CMV) immediate early promoter, the simian virus 40 (SV40) (e.g., early or late) promoter, the Moloney murine leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, the herpes simplex virus (HSV) (thymidine kinase) promoter, the SV40 / CD43 promoter, the spleen lesion-forming virus (SFFV) promoter, the elongation factor 1-α (EFlα) short promoter (without introns), the intron-containing EFlα long promoter, the ubiquitin C (UBC) promoter, the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and the myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al., Journal of Virology). 2003;77(17): 9439-9450).

[0325] Illustrative examples of general expression control sequences applicable to the specific implementation schemes considered herein include those sequences that contain the polynucleotide sequences listed in Table 7.

[0326] Table 7

[0327] In a specific implementation, the polynucleotide includes one or more cell type or tissue-specific expression control sequences. In a specific implementation, the cell type-specific expression control sequence is targeted at immune effector cells. In a specific implementation, the cell type-specific expression control sequence is a T cell-specific promoter, an NK cell-specific promoter, an NKT cell-specific promoter, or a mucosa-associated invariant T (MAIT) cell promoter.

[0328] In the specific implementation scheme, the cell type-specific expression control sequence is selected from: distal lymphocyte protein tyrosine kinase (LCK) promoter (Brenner et al., Proc. Natl. Acad. Sci. USA 99:2936-2941 (2002)), CD3δ promoter (Ji et al., J Biol Chem. 277(49):47898-906 (2002)), CD4 gene promoter (Salmon et al., Proc. Natl. Acad. Sci. USA 90:7739 (1993), CD2 promoter (Greaves et al., Cell 56:979-86 (1989)) and TCF7 promoter (van de Wetering et al. J. of Bio. Chem.267: 8530-8536 (1992)).

[0329] In specific implementation schemes, the expression of polynucleotide sequences can be regulated by integrating post-transcriptional regulatory elements into vectors. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids, such as the prairie hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73: 2886); the post-transcriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); and so on (Liu et al., 1995, Genes Dev., 9:1766).

[0330] Illustrative examples of post-transcriptional control sequences applicable to the specific implementation schemes considered herein include sequences comprising the polynucleotide sequences shown in Table 8.

[0331] Table 8

[0332] In a specific embodiment, the vector contains or encodes (in the case of an RNA vector such as a retroviral vector) an MNDU3 promoter (e.g., SEQ ID NO: 950), said promoter being operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing any of the posttranscriptional regulatory elements shown in SEQ ID NO: 945-947. In a specific embodiment, the vector comprises or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an anti-BCMA antibody or its antigen-binding fragment containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141, and optionally a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947. In another specific embodiment, the vector comprises or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an amino acid sequence shown in any one of SEQ ID NO: 165-860, and optionally a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947. In a specific embodiment, the vector comprises or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a signal peptide and a polynucleotide encoding a chimeric antigen receptor, wherein the signal peptide comprises any one of the amino acid sequences described in SEQ ID NO: 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, and 873, and the chimeric antigen receptor comprises any one of the amino acid sequences described in SEQ ID NO: 165-860, and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in SEQ ID NO: 945-947. In a specific embodiment, the vector contains or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an amino acid sequence represented by any one of SEQ ID NOs: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837; and optionally a polynucleotide comprising a post-transcriptional regulatory element represented by any one of SEQ ID NOs: 945-947.In a specific embodiment, the vector comprises or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a signal peptide and a polynucleotide encoding a chimeric antigen receptor, the signal peptide comprising the amino acid sequence shown in SEQ ID NO: 861, the chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837; and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947. In a specific embodiment, the vector comprises or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising the polynucleotide sequence shown in any one of SEQ ID NO: 905-944, and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947. In another specific embodiment, the vector comprises or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the signal peptide comprising the polynucleotide sequence shown in SEQ ID NO: 904, the chimeric antigen receptor comprising the polynucleotide sequence shown in any one of SEQ ID NO: 905-924, and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947. In a specific implementation, the vector contains or encodes an MNDU3 promoter operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor comprising a polynucleotide sequence shown in any one of SEQ ID NO: 925-944, and optionally a polynucleotide comprising a posttranscriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0333] In a specific embodiment, the vector contains or encodes (in the case of an RNA vector such as a retroviral vector) an EF1α promoter (e.g., SEQ ID NO: 949), said promoter being operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any of SEQ ID NO: 945-947. In a specific embodiment, the vector comprises or encodes an EF1α promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141, and optionally a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947. In another specific embodiment, the vector comprises or encodes an EF1α promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an amino acid sequence shown in any one of SEQ ID NO: 165-860, and optionally a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947. In a specific embodiment, the vector comprises or encodes an EF1α promoter operatively linked to a polynucleotide encoding a signal peptide and a polynucleotide encoding a chimeric antigen receptor, wherein the signal peptide comprises any one of the amino acid sequences described in SEQ ID NO: 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, and 873, and the chimeric antigen receptor comprises any one of the amino acid sequences described in SEQ ID NO: 165-860, and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in SEQ ID NO: 945-947. In a specific embodiment, the vector contains or encodes an EF1α promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising an amino acid sequence represented by any one of SEQ ID NOs: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837; and optionally a polynucleotide comprising a posttranscriptional regulatory element represented by any one of SEQ ID NOs: 945-947.In a specific embodiment, the vector comprises or encodes an EF1α promoter operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the signal peptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, and 873, the chimeric antigen receptor comprising an amino acid sequence represented by any one of SEQ ID NOs: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837; and optionally a polynucleotide comprising a post-transcriptional regulatory element represented by any one of SEQ ID NOs: 945-947. In a specific embodiment, the vector comprises or encodes an EF1α promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising the polynucleotide sequence shown in any one of SEQ ID NO: 905-924, and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947. In another specific embodiment, the vector comprises or encodes an EF1α promoter operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the signal peptide comprising the polynucleotide sequence shown in SEQ ID NO: 904, the chimeric antigen receptor comprising the polynucleotide sequence shown in any one of SEQ ID NO: 905-924, and optionally a polynucleotide comprising a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947. In a specific embodiment, the vector contains or encodes an EF1α promoter operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising a polynucleotide sequence shown in any one of SEQ ID NO: 925-944, and optionally a polynucleotide comprising a posttranscriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0334] In some implementations, efficient expression of polynucleotides can also be increased by using sequences that enhance translation efficiency, such as by increasing mRNA ribosome binding or increasing mRNA stability. In some implementations, the polynucleotide encoding the chimeric antigen receptor contains a short recognition sequence, namely the Kozak sequence, which greatly facilitates the initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, Cell. 44:283-92 (1986), and Kozak, Nucleic Acids Res. 15:8125-48 (1987)).

[0335] Elements that guide the efficient termination and polyadenylation of heterologous nucleic acid transcripts can also enhance heterologous gene expression. Transcription termination signals are typically located downstream of polyadenylation signals. In specific implementations, the vector contains a polyadenylated sequence located at the 3' end of the sequence to be transcribed and / or expressed. "Polyadenylation (or poly(A) signal)" refers to a DNA sequence that directs RNA polymerase II to terminate nascent RNA transcripts and polyadenylate them. Polyadenylation signals can contribute to improved translation efficiency by adding a poly(A) tail to the 3' end of the coding sequence, thereby promoting mRNA stability. Poly(A) signals in RNA guide cleavage and polyadenylation. The core poly(A) signal in mammalian precursor mRNA has two recognition elements flanking cleavage-polyadenylation sites. Typically, the nearly invariant AAUAAA hexamer is located 20–50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled with the addition of up to 250 adenosines to the 5' cleavage product. In a specific embodiment, the core poly(A) signal is an ideal poly(A) signal (e.g., AATAAA, ATTAAA, AGTAAA). In a specific embodiment, the poly(A) signal is the SV40 poly(A) signal, bovine growth hormone poly(A) signal (BGHpA), rabbit β-globin poly(A) signal (rβgpA), variants thereof, or other suitable heterologous or endogenous poly(A) signals known in the art. In a specific embodiment, the poly(A) signal is synthetic.

[0336] In a specific embodiment, the polynucleotide comprises or encodes a promoter operatively encoding a polynucleotide sequence encoding a chimeric antigen receptor comprising a signal peptide isolated from a polypeptide selected from CD8α, mouse IgGκ, human IgGk, CD33, tPA, SEAP, hGM-CSF, Gaussian luciferase, CSF2R, B2M, and CD80, wherein the signal peptide is subsequently cleaved from the translated chimeric antigen receptor. In another specific embodiment, the polynucleotide comprises or encodes a promoter operatively linked to a polynucleotide sequence encoding a chimeric antigen receptor comprising a signal peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 861-873. An illustrative example of a polynucleotide encoding a signal peptide is given in SEQ ID NO: 904 (ATGGCTCTTCCCGTAACAGCCCTTTTGTTGCCCCTTGCACTCCTTCTGCATGCAGCACGACCG).

[0337] In a specific implementation, the polynucleotide includes one or more miR target sequences inserted into the 5' UTR, introns, and / or 3' UTR to restrict expression in unwanted or off-target cell types.

[0338] In some embodiments, the polynucleotide contains an inducible suicide gene to reduce the risk of direct toxicity and / or uncontrolled proliferation. In some embodiments, the suicide gene is caspase-8 or caspase-9. Caspase-9 can be activated using a specific chemical dimerization inducer (CID).

[0339] In some implementations, the polynucleotide contains a gene or gene segment that, when introduced into a cell, makes the cell susceptible to negative selection. Negative selection suitable for a particular implementation includes, but is not limited to, the HSV-TK gene that confers ganciclovir sensitivity; cellular hypoxanthine phosphoribosyltransferase (HPRT) genes, cellular adenine phosphoribosyltransferase (APRT) genes, and bacterial cytosine deaminases.

[0340] In some embodiments, the polynucleotide contains a gene or gene segment that, when introduced into a cell, makes the cell susceptible to positive selection. Positive selection genes applicable to the specific embodiments considered herein include, but are not limited to: the hygromycin B phosphotransferase gene (hph) confers resistance to hygromycin B; the aminoglycoside phosphotransferase gene (neo or aph) from Tn5 encoding resistance to the antibiotic G418; the dihydrofolate reductase (DHFR) gene; the adenosine deaminase gene (ADA); and the multidrug resistance (MDR) gene.

[0341] Table 9 lists the SEQ ID NO. and associated nucleic acid sequences encoding chimeric antigen receptor components and chimeric antigen receptors, as well as the corresponding amino acid SEQ ID NO (AA SEQ ID NO.) encoded by the nucleic acid sequences.

[0342] Table 9

[0343] In a specific embodiment, the vector comprises a polynucleotide encoding a polypeptide of the kind considered herein. In a specific embodiment, the polypeptide is selected from antibodies, antigen-binding fragments of antibodies, bispecific antibodies, BiTEs, and chimeric antigen receptors.

[0344] In a specific embodiment, the vector comprises a polynucleotide encoding a polypeptide, said polypeptide comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acid sequences shown in SEQ ID NO: 11-144.

[0345] In a specific embodiment, the vector comprises a polynucleotide encoding a bispecific antibody, the bispecific antibody comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acid sequences shown in SEQ ID NO:11-144; and optionally a polypeptide linker and an anti-CD3 antibody.

[0346] In a specific embodiment, cells (e.g., immune effector cells) are modified to express a polypeptide (e.g., a chimeric antigen receptor), said polypeptide comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acid sequences shown in SEQ ID NO: 11-144.

[0347] In specific embodiments, the polynucleotides considered herein, or vectors containing or encoding such polynucleotides, are introduced into cells, such as immune effector cells. In specific embodiments, non-viral vectors containing polynucleotides are introduced into cells. Illustrative examples of non-viral vectors include, but are not limited to: autonomously replicating sequences; plasmids; phage particles; granules; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages, such as λ phage or M13 phage; and transposon elements, including but not limited to piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and derivatives thereof.

[0348] In a specific implementation, a viral vector containing multiple nucleotides is introduced into the cell. Illustrative examples of viral vectors include, but are not limited to: adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, rhabdovirus (e.g., rabies virus, vesicular virus) vectors, paramyxovirus (e.g., Hennipa virus, measles virus, respiratory virus, rubella virus) vectors, herpes simplex virus (e.g., HSV-1, HSV-2) vectors, vaccinia virus vectors, and retroviral vectors, preferably lentiviral vectors (LVV).

[0349] A "viral vector" is a nucleic acid molecule derived from a viral genome used to transfer or deliver another nucleic acid into a cell. Viral vectors are based on and derived from the viral genome, engineered to remove viral accessory proteins but retain elements required for packaging, reverse transcription, and integration. In a preferred embodiment, the viral vectors considered herein comprise a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding a polypeptide comprising an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a chimeric antigen receptor (CAR).

[0350] In specific embodiments, the adenoviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding a polypeptide containing an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a CAR. High-capacity adenoviral vectors (HC-Ad) (third generation) retain only the short non-coding regions of the Ad genome (ITR and ψ signal), allowing the vector tp to carry a large polynucleotide payload (approximately 37 kb).

[0351] In specific embodiments, the AAV vector comprises a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding a polypeptide containing an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a CAR. The recombinant AAV (rAAV) vector is maintained primarily in free form and has a polynucleotide payload capacity of approximately 4.7 kb. The rAAV vector typically consists of at least a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The rAAV vector may contain an ITR from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. The construction of rAAV vectors and the production and purification of AAV have been disclosed in, for example, U.S. Patent Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058 and 8,784,799, each of which is incorporated herein by reference in its entirety.

[0352] In specific implementations, HSV vectors contain a polynucleotide that includes or encodes a promoter operatively linked to a polynucleotide encoding a polypeptide containing an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a CAR. HSV vectors are relatively large, for example, up to 152 kb. Typically, HSV vectors are designed to be replication-deficient; furthermore, one or more essential or non-essential HSV genes are removed from the vector backbone to make room for the polynucleotide payload. Most replication-deficient HSV vectors contain deletions to remove one or more immediate early, early, or late HSV genes, thereby preventing replication. An advantage of HSV vectors is their ability to enter a latent phase, leading to long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA inserts up to 25 kb. HSV-based vectors are described in, for example, U.S. Patent Nos. 5,837,532, 5,846,782 and 5,804,413 and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637 and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0353] In a specific embodiment, the retroviral or lentiviral vector comprises a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding a polypeptide comprising an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a CAR. In another specific embodiment, the recombinant particle comprises two copies of the vector, namely, genomic RNA containing a backbone sequence from a retroviral genome, such as a lentiviral genome.

[0354] In various implementations, the retroviral vector is engineered or derived from a retroviral genome selected from: α-retroviral genome, β-retroviral genome, γ-retroviral genome, δ-retroviral genome, or foam virus genome (e.g., ε-retroviral genome, simian foam virus genome, bovine foam virus genome, equine foam virus genome, feline foam virus genome, and prosimian foam virus genome).

[0355] In a specific implementation, the retroviral vector comprises 5' LTR and 3' LTR, which are each isolated, obtained, or derived from a retroviral genome selected from: α-retroviral genome, β-retroviral genome, γ-retroviral genome, δ-retroviral genome, ε-retroviral genome, and foam virus genome.

[0356] Illustrative examples of α-retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, avian leukosis virus, avian cancermilhill virus 2, avian myeloblastosis virus, avian myeloblastomavirus 29, avian sarcoma virus CT10, Fujinami sarcoma virus, Rous sarcoma virus, UR2 sarcoma virus, and Y73 sarcoma virus.

[0357] Illustrative examples of β-retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, mouse mammary tumor virus, Jaagsiekte sheep retrovirus, long-tailed langur virus, Mason-Pfizer monkey virus, and squirrel monkey retrovirus (SMRV).

[0358] Illustrative examples of delta retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, bovine leukemia virus, primate T-lymphocyte virus 1, primate T-lymphocyte virus 2, primate T-lymphocyte virus 3, and primate T-lymphocyte virus 4.

[0359] Illustrative examples of ε-retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, pikeperch cutaneous sarcoma virus, pikeperch epidermal proliferative virus 1, and pikeperch epidermal proliferative virus 2.

[0360] Illustrative examples of gamma retroviruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, baboon endogenous virus (BaEV), chicken syncytial virus, feline endogenous virus (e.g., RD114), feline leukemia virus (FeLV), Finkel-Biskis-Kinggins mouse sarcoma virus, Gardner-Arnstein feline sarcoma virus, gibberish leukemia virus (GALV), guinea pig type C tumor virus, Hardy-Zuckerman feline sarcoma virus, Harvey mouse sarcoma virus, Kersten mouse sarcoma virus, koala retrovirus, murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus, porcine endogenous virus (PERV), porcine type C tumor virus, reticuloendothelial proliferation virus (REV), Snyder-Tyron feline sarcoma virus, Tregue duck spleen necrosis virus, viper retrovirus, heterophilic murine leukemia virus-associated virus (XMRV), and trichome sarcoma virus.

[0361] Illustrative examples of foam viruses from which retroviral vectors can be isolated, obtained, or derived include, but are not limited to, simian foam virus, bovine foam virus, equine foam virus, feline foam virus, human foam virus (HFV), and brown baby monkey prosimian foam virus.

[0362] In various embodiments, the lentiviral vector (lentiviral vector) is engineered or derived from a lentiviral genome. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); viscenario-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In specific embodiments, the lentiviral vector is derived from the HIV viral genome, preferably the HIV-1 or HIV-2 viral genome, more preferably the HIV-1 viral genome (i.e., preferably HIV-1 cis-acting sequence elements).

[0363] In various embodiments, the lentivirus comprises two copies of a lentiviral vector-based RNA genome containing a 5' long terminal repeat (LTR) with an R region and a U5 region; a Psi(ψ) packaging signal; cPPT / FLAP, an output element; a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding a polypeptide containing an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a CAR; a 3' LTR containing a U3 region and an R region; optional WPRE or HPRE; a polyadenylation signal; and a poly(A) tail.

[0364] As used in this article, the term "long terminal repeat (LTR)" refers to an element located at the polynucleotide end of a retrovirus, which is a homologous repeat containing U3, R, and U5 regions in its natural sequence background. LTRs typically provide functions crucial for retroviral gene expression (e.g., initiation, startup, and polyadenylation of gene transcripts) and viral replication. LTRs contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. Viral LTRs are divided into three regions, designated U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region, containing polyadenylation signals. The R (repeat) region is flanked by the U3 and U5 regions. Transfer plasmids used to package vector genomes contain a 5' LTR containing U3, R, and / or U5 regions and a 3' LTR containing U3, R, and / or U5 regions. The 5′ LTR is located near sequences essential for genome reverse transcription (tRNA primer binding site) and sequences essential for efficient packaging of viral RNA into particles (Psi "Ψ" site). The genome packaged in the particle from a retroviral vector contains a 5′ LTR with R and U5 regions and a 3′ LTR with U3 and R regions. The retroviral vector-based genome is reverse transcribed and integrated into the host cell genome as a pre-vector. Through reverse transcription of the retroviral vector genome and second-strand synthesis, the pre-vector contains two copies of the 3′ LTR, one copy replacing both the 5′ LTR and the 3′ LTR.

[0365] The “TAR” element used in this article refers to the “transactivation response” genetic element located in the R region of the lentiviral vector LTR. This element interacts with the lentiviral transactivator (tat) genetic element to enhance lentiviral vector genome replication. In third-generation lentiviral vectors, this element is typically absent because the lentiviral transfer vector contains the 5'LTR U3 region replaced by a heterologous promoter.

[0366] As used in this article, the "R region" refers to the region in the LTR that begins at the start of the capping group (i.e., the start of transcription) and ends before the start of the polyA sequence. The R region is also defined as flanking the U3 and U5 regions. The R region plays a role in reverse transcription, allowing newly generated DNA to be transferred from one end of the genome to the other.

[0367] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located within the retroviral genome, which is required for the insertion of viral RNA into the viral capsid or particle; see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101–2109. Several retroviral vectors use a minimal packaging signal (also known as a psi [ψ] or [ψ+] sequence) required for viral genome capsid formation. Therefore, as used herein, the terms "packaging sequence," "packaging signal," "psi," and the symbol "Ψ" refer to the non-coding sequence required for the encapsulation of the retroviral RNA strand during viral particle formation.

[0368] As used herein, a “FLAP element” or “cPPT / FLAP” refers to a nucleic acid whose sequence includes the central polypurine bundle and central termination sequence (cPPT and CTS) of a lentivirus (e.g., HIV-1 or HIV-2). “FLAP element” and “cPPT / FLAP” are used interchangeably to refer to the aforementioned FLAP element. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, positive-strand DNA begins at the central polypurine sequence (cPPT) and terminates at the central termination sequence (CTS), forming a triple-stranded DNA structure: the HIV-1 central DNA lobe. While not wishing to be bound by any particular theory, the DNA lobe can serve as a cis-activity determinant of lentiviral genome nuclear input and / or can increase viral titer.

[0369] As used herein, the term “output element” refers to a cis-acting posttranscriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm. Examples of RNA output elements include, but are not limited to, human immunodeficiency virus (HIV) rev response elements (RREs) (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), marmot hepatitis virus posttranscriptional regulatory elements (WPREs), and hepatitis B virus posttranscriptional regulatory elements (HPREs).

[0370] By integrating post-transcriptional regulatory elements, effective polyadenylation signals, and optional transcription termination signals into vectors, the expression of heterologous sequences in viral vectors can be increased. Various post-transcriptional regulatory elements, such as WPRE and HPRE, can increase the expression of heterologous nucleic acids on proteins.

[0371] Lentiviral vectors may contain one or more safety enhancements to reduce the risks of replication, insertional mutagenesis, and off-target transduction and / or expression. In specific embodiments, lentiviral vectors contain one or more of the following safety enhancements: one or more modifications to the 5' and 3' LTRs, cell or tissue-specific expression control sequences, such as promoters, enhancers, or miRNA target sequences. As used herein, "modified LTR" refers to the addition, deletion, or substitution of one or more nucleotides in the native HIV-1 5' LTR and / or 3' LTR. Those skilled in the art will be able to determine whether an LTR has been modified by comparison with a reference LTR.

[0372] As used herein, a “self-inactivating” (SIN) vector refers to a replication-defective vector, such as a retroviral or lentiviral vector, in which the right-hand (3ˊ) LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Self-inactivation is achieved by deleting the U3 region of the 3ˊ LTR of the lentiviral vector transfer plasmid, thereby removing the LTR TATA box (e.g., deletion from -418 to -18) without significantly reducing the titer.

[0373] Additional safety enhancements are provided by replacing the U3 region of the 5ˊ LTR with a heterologous promoter to drive transcription of the viral genome during the production of recombinant viral particles. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0374] In specific implementation schemes, lentiviral vectors are engineered to integrate into the host cell genome.

[0375] In some embodiments, the lentiviral vector is engineered to be integration-deficient, free, and not integrated into the cellular genome. As used herein, the term "integration-deficient lentivirus" or "IDLV" refers to a lentivirus lacking the integrase's ability to integrate the viral vector into the host cell genome. Viral vectors lacking integration capability have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety. Illustrative mutations that can reduce HIV-1 integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, K156E, K156A, E157A, K159 E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D2 02A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H. In a specific implementation, the HIV-1 integration-deficient integrase includes mutations in D64V, D161I, D116A, E152G, or E152A; mutations in D64V, D116A, and E152G; mutations in D64V, D116A, and E152A; or a D64V mutation.

[0376] H. cells

[0377] In a specific embodiment, a polynucleotide encoding the polypeptide described herein is introduced into a cell, such as an immune effector cell. In a specific embodiment, the cell (e.g., an immune effector cell) is modified to express a polypeptide comprising an anti-BCMA binding protein, an anti-BCMA binding protein (e.g., an anti-BCMA antibody or its antigen-binding fragment), an anti-BCMA-anti-CD3 bispecific antibody, or a CAR.

[0378] In a specific embodiment, cells (e.g., immune effector cells) are modified to express the polypeptide of consideration, said polypeptide comprising an anti-BCMA binding protein, such as an anti-BCMA antibody or its antigen-binding fragment, an anti-BCMA-anti-CD3 bispecific antibody, or a CAR comprising any one of the amino acid sequences shown in SEQ ID NO: 11-144, or a CAR comprising any one of the amino acid sequences shown in SEQ ID NO: 165-860.

[0379] "Immune effector cells" refer to cells in the immune system that possess one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, ADCC and / or CDC induction). Illustrative types of immune effector cells considered in specific embodiments include, but are not limited to, T lymphocytes, dendritic cells (DCs), Treg cells, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages. The terms "T cell" or "T lymphocyte" are recognized in the art and, in specific embodiments, are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, and / or activated T lymphocytes. Illustrative examples of T lymphocytes suitable for specific embodiments include, but are not limited to, cytotoxic T cells (CTLs; CD8+). + T cells, TILs, helper T cells (HTL; CD4) + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subset of T cells with effector functions. In a specific embodiment, the cells include α-β T cells. In a specific embodiment, the cells include γδ T cells.

[0380] In a specific implementation, immune effector cells include natural killer (NK) cells. NK cells do not express T cell antigen receptor (TCR), CD3, or surface immunoglobulin (Ig) B cell receptor, but in humans they typically express the surface markers CD16 (FcyRIII) and CD56.

[0381] In the specific implementation plan, immune effector cells include natural killer T (NKT) cells.

[0382] In a specific implementation, a polynucleotide encoding the polypeptide considered herein is introduced into a progenitor cell of an immune effector cell, subsequently inducing its differentiation or differentiation into one or more immune effector cells. In a specific implementation, the progenitor cells of the immune effector cells include hematopoietic stem cells (HSCs); said hematopoietic stem cells contain CD34-containing progenitor cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood. + Within the cell population, they can naturally differentiate into mature immune effector cells, or can be induced to differentiate into mature immune effector cells.

[0383] I. Compositions and Formulations

[0384] The compositions considered herein comprise one or more antibodies or antigen-binding fragments thereof, bispecific antibodies, antibody conjugates, peptides, fusion peptides, chimeric antigen receptors, polynucleotides, carriers, and / or in vitro modified immune effector cells.

[0385] In a specific embodiment, the composition comprises one or more polynucleotides and / or polypeptides.

[0386] In a specific embodiment, the composition comprises a polynucleotide containing or encoding a promoter operatively linked to one or more polynucleotides encoding one or more anti-BCMA binding proteins, such as anti-BCMA antibodies or their antigen-binding fragments, anti-BCMA-anti-CD3 bispecific antibodies, or CARs.

[0387] In a specific embodiment, the composition comprises a vector containing a polynucleotide that contains or encodes a promoter operatively linked to a polynucleotide encoding an amino acid sequence shown in any one of SEQ ID NO: 165-860 or a polynucleotide sequence shown in any one of SEQ ID NO: 905-944.

[0388] In the specific implementation scheme, the composition is a pharmaceutical composition. A "pharmaceutical composition" means a composition formulated into a pharmaceutically acceptable or physiologically acceptable solution for administration alone or in combination with one or more other therapeutic modalities to cells or subjects.

[0389] "Pharmaceutical acceptable" means that the molecular entity and composition, when taken orally to a human, will not produce excessive toxicity, irritation, allergic reactions or other problems or complications, and has a reasonable benefit / risk ratio.

[0390] In specific embodiments, the composition comprises a pharmaceutically acceptable carrier and the recombinant particles considered herein. “Pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, mediator, etc., that is physiologically compatible with the polypeptide, polynucleotide, or carrier and is compatible with human administration, including but not limited to pharmaceutically acceptable cell culture media, Duchenne phosphate-buffered saline (PBS), Ringer's solution, 5% glucose aqueous solution (D5W), and physiological saline (0.9% NaCl).

[0391] In specific embodiments, the composition comprises a polypeptide, a polynucleotide, or a carrier and a pharmaceutically acceptable carrier, suitable for intravenous or parenteral administration, such as intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrasheath, intramuscular, and intramedullary administration and formulation.

[0392] In specific embodiments, the composition is substantially free of mycoplasma, endotoxins, and microbial contamination. "Substantially free of endotoxins" means that the endotoxin content per dose of cells is below the U.S. Food and Drug Administration (FDA) allowable standard for biological products, which is a total endotoxin content of 5 EU per kilogram of body weight per day. For an average person weighing 70 kg, the total endotoxin content per dose of cells is 350 EU. In specific embodiments, the composition considered herein contains approximately 0.5 EU / mL to approximately 5.0 EU / mL, or approximately 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 EU / mL, or 5.0 EU / mL.

[0393] In specific embodiments, the compositions considered herein are intended for the treatment of cancer, GVHD, infectious diseases, autoimmune diseases, inflammatory diseases, or immunodeficiency. In specific embodiments, the compositions comprise the recombinant particles described herein, as well as one or more cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other active and adjuvant agents, which may be used alone or in combination.

[0394] Those skilled in the art will understand that the specific embodiments considered herein may include other formulations, such as those well known in the pharmaceutical field and described in, for example... Remington: The Science and Practice of Pharmacy , Volume I and Volume II. 23rd edition. Edited by Adeboye Adejare. Academic Press, 2020. The formulation of this literature is incorporated herein by reference in its entirety.

[0395] J. List of Implementation Schemes

[0396] Implementation Scheme 1: An antibody or its antigen-binding fragment, comprising: (a) A heavy chain variable region (VH), comprising CDRH1, CDRH2, and CDRH3 of the antibody or its antigen-binding fragment as shown in Table 1; a peptide linker; and a light chain variable region (VL), comprising CDRL1, CDRL2, and CDRL3 of the antibody or its antigen-binding fragment as shown in Table 1; or (b) VHH domains of CDRH1, CDRH2 and CDRH3 containing the antibodies or their antigen-binding fragments shown in Table 1.

[0397] Implementation Scheme 2: The antibody or its antigen-binding fragment as described in Implementation Scheme 1, wherein: (a) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 12, 13 and 14, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 16, 17 and 18; (b) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 22, 23 and 24, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 26, 27 and 28; (c) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 32, 33 and 34, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 36, 37 and 38; (d) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 42, 43 and 44, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 46, 47 and 48; (e) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 52, 53 and 54, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 56, 57 and 58; (f) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 62, 63 and 64, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 66, 67 and 68; (g) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 72, 73 and 74, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 76, 77 and 78; (h) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 82, 83 and 84, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 86, 87 and 88; (i) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 92, 93 and 94, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 96, 97 and 98; (j) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 102, 103 and 104; (k) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 106, 107 and 108; (l) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 110, 111 and 112; (m) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 114, 115 and 116; (n) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 118, 119 and 120; (o) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 122, 123 and 124; (p) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 126, 127 and 128; (q) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 130, 131 and 132; (r) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 134, 135 and 136; (s) The VHH domain contains CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 138, 139, and 140; or (t) The VHH domain contains CDRH1, CDRH2, and CDRH3, which contain the amino acid sequences shown in SEQ ID NO: 142, 143, and 144.

[0398] Implementation Scheme 3: An antibody or its antigen-binding fragment as described in Implementation Scheme 1 or Implementation Scheme 2, wherein: (a) VH contains the amino acid sequence shown in SEQ ID NO: 11, and VL contains the amino acid sequence shown in SEQ ID NO: 15; (b) VH contains the amino acid sequence shown in SEQ ID NO: 21, and VL contains the amino acid sequence shown in SEQ ID NO: 25; (c) VH contains the amino acid sequence shown in SEQ ID NO: 31, and VL contains the amino acid sequence shown in SEQ ID NO: 35; (d) VH contains the amino acid sequence shown in SEQ ID NO: 41, and VL contains the amino acid sequence shown in SEQ ID NO: 45; (e) VH contains the amino acid sequence shown in SEQ ID NO: 51, and VL contains the amino acid sequence shown in SEQ ID NO: 55; (f) VH contains the amino acid sequence shown in SEQ ID NO: 61, and VL contains the amino acid sequence shown in SEQ ID NO: 65; (g) VH contains the amino acid sequence shown in SEQ ID NO: 71, and VL contains the amino acid sequence shown in SEQ ID NO: 75; (h) VH contains the amino acid sequence shown in SEQ ID NO: 81, and VL contains the amino acid sequence shown in SEQ ID NO: 85; (i) VH contains the amino acid sequence shown in SEQ ID NO: 91, and VL contains the amino acid sequence shown in SEQ ID NO: 95; or (a) The VHH domain contains any one of the amino acid sequences shown in SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

[0399] Implementation Scheme 4: The antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes 1 to 3, wherein the polypeptide linker is selected from: TGEKP (SEQ ID NO: 2); (GGGGS) nWhere n = 1, 2, 3, 4 or 5 (SEQ ID NO: 3 and 976-979); EGKSSGSGSESKVD (SEQ ID NO: 4); KESGSVSSEQLAQFRSLD (SEQ ID NO: 5); LRQRDGERP (SEQ ID NO: 6); LRQKDGGGSERP (SEQ ID NO: 7); LRQKD(GGGS)2ERP (SEQ ID NO: 8), GETSTGSGGSGGSGGAD (SEQ ID NO: 9) and GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 10) and their amino acid sequence variants containing 95% identical amino acid sequences.

[0400] Implementation Scheme 5: An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence represented by any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

[0401] Implementation Scheme 6: A bispecific antibody comprising an antibody or an antigen-binding fragment thereof from any one of Implementation Schemes 1 to 5.

[0402] Implementation Scheme 7: The bispecific antibody as described in Implementation Scheme 6 further comprises an anti-CD3 antibody that binds to CD3δ, CD3ε, CD3γ or CD3ζ.

[0403] Implementation Scheme 8: An antibody conjugate comprising an antibody or an antigen-binding fragment thereof from any one of Implementation Schemes 1 to 5.

[0404] Implementation Scheme 9: An antibody conjugate as described in Implementation Scheme 8, wherein the antigen or its antigen-binding fragment is conjugated to a cytotoxic agent.

[0405] Implementation Scheme 10: An antibody conjugate as described in Implementation Scheme 8 or Implementation Scheme 9, wherein: (a) The cytotoxic agent is selected from the following toxins: saponins, diphtheria toxin, Pseudomonas exotoxin A, ricin A chain derivatives, small molecule toxins and combinations thereof; (b) The cytotoxic agent is a radioactive isotope selected from the following: 131I, 90Y, 177Lu, 188Re, 67Cu, 213Bi, 211At and 227Ac; (c) The cytotoxic agent is selected from the following RNA polymerase II inhibitors and / or RNA polymerase III inhibitors: amatoxins, α-amaminine, β-amaminine, γ-amaminine, ε-amaminine, amatoxins, amatoxin amide, amatoxin non-toxic cyclic peptide, amatoxin acid, and any functional fragments, derivatives, or analogs thereof; or (d) The cytotoxic agent is selected from the following DNA damaging agents: anti-tubulin agents, DNA cross-linking agents, DNA alkylating agents and mitotic disruptors.

[0406] Implementation Scheme 11: A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof of any one of Implementation Schemes 1 to 5; a spacer domain; a transmembrane domain and one or more intracellular signal transduction domains.

[0407] Implementation Scheme 12: The CAR as described in Implementation Scheme 11, wherein the spacer domain comprises a hinge domain selected from the following or a fragment thereof: CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge and IgG4 hinge.

[0408] Implementation Scheme 13: The CAR as described in Implementation Scheme 11 or Implementation Scheme 12, wherein the spacer domain comprises an amino acid sequence shown in any one of SEQ ID NO: 145, 146, 147, 148, 149 and 150 or an amino acid sequence that is at least 95% identical to it.

[0409] Implementation Scheme 14: The CAR as described in any one of Implementation Schemes 11 to 13, wherein the transmembrane domain is isolated from or derived from a polypeptide selected from: α, β, γ or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death 1 (PDCD1).

[0410] Implementation Scheme 15: CAR as described in any one of Implementation Schemes 11 to 14, wherein the transmembrane domain comprises an amino acid sequence shown in any one of SEQ ID NO: 151, 152, 153, 154, 155, 156 and 157 or an amino acid sequence that is at least 95% identical thereto.

[0411] Implementation Scheme 16: The CAR of any one of Implementation Schemes 11 to 15, wherein the one or more intracellular signal transduction domains comprise major signal transduction domains isolated or derived from a polypeptide selected from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0412] Implementation Scheme 17: CAR as described in any one of Implementation Schemes 11 to 16, wherein the one or more intracellular signal transduction domains comprise a major signal transduction domain separated from CD3ζ.

[0413] Implementation Scheme 18: The CAR as described in Implementation Scheme 17, wherein the primary signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical thereto.

[0414] Implementation Scheme 19: CAR as described in any one of Implementation Schemes 11 to 18, wherein the one or more intracellular signal transduction domains comprise co-stimulatory signal transduction domains isolated from or derived from a polypeptide selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

[0415] Implementation Scheme 20: CAR as described in any one of Implementation Schemes 11 to 19, wherein the one or more intracellular signal transduction domains comprise a co-stimulatory signal transduction domain, the co-stimulatory signal transduction domain comprising an amino acid sequence shown in any one of SEQ ID NO: 159, 160, 161, 162, 163 and 164 or an amino acid sequence that is at least 95% identical thereto.

[0416] Implementation Scheme 21: A CAR comprising: an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, 90, 101 or 117; a spacer domain comprising an amino acid sequence shown in any one of SEQ ID NO: 145, 146 and 148, or an amino acid sequence at least 95% identical thereto; a transmembrane domain comprising an amino acid sequence shown in SEQ ID NO: 151 or 153; and one or more intracellular signal transduction domains comprising a co-stimulatory signal transduction domain comprising an amino acid sequence shown in any one of SEQ ID NO: 159, 160 and 162, or an amino acid sequence at least 95% identical thereto; and further comprising a major signal transduction domain comprising an amino acid sequence shown in SEQ ID NO: 158, or an amino acid sequence at least 95% identical thereto.

[0417] Implementation Scheme 22: A CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 165-860.

[0418] Implementation Scheme 23: A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282 and 283.

[0419] Implementation Scheme 24: A CAR comprising an amino acid sequence represented by any one of SEQ ID NO: 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379 and 380.

[0420] Implementation Scheme 25: A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 and 452.

[0421] Implementation Scheme 26: A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547 and 548.

[0422] Implementation Scheme 27: A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619 and 620.

[0423] Implementation Scheme 28: A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715 and 716.

[0424] Implementation Scheme 29: The CAR described in any one of Implementation Schemes 11 to 28 further comprises a signal peptide.

[0425] Implementation Scheme 30: CAR as described in Implementation Scheme 29, wherein the signal peptide comprises an amino acid sequence represented by any one of SEQ ID NO: 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872 and 873.

[0426] Implementation Scheme 31: A polynucleotide encoding CAR comprising the polynucleotide sequence shown in any one of SEQ ID NO: 905-924.

[0427] Implementation Scheme 32: A polynucleotide encoding a signal peptide and CAR, comprising the polynucleotide sequence shown in any one of SEQ ID NO: 925-944.

[0428] Implementation Scheme 33: A polynucleotide encoding an antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes 1 to 5, a bispecific antibody as described in Implementation Scheme 6 or Implementation Scheme 7, an antibody conjugate as described in any one of Implementation Schemes 8 to 10, or a CAR as described in any one of Implementation Schemes 11 to 30.

[0429] Implementation Scheme 34: A polynucleotide encoding or containing a promoter operatively linked to a polynucleotide of any one of Implementation Schemes 31 to 33.

[0430] Implementation Scheme 35: The polynucleotide as described in Implementation Scheme 34, wherein the promoter comprises the polynucleotide sequence shown in any one of SEQ ID NO: 980, 981, 982, 983, 984 and 985.

[0431] Implementation Scheme 36: The polynucleotide as described in Implementation Scheme 34 or Implementation Scheme 35 further comprises a post-transcriptional response element.

[0432] Implementation Scheme 37: The polynucleotide of Implementation Scheme 36, wherein the post-transcriptional response element comprises the polynucleotide sequence shown in any one of SEQ ID NO: 945, 946 and 947.

[0433] Implementation Scheme 38: A DNA comprising the polynucleotide sequence of any one of Implementation Schemes 31 to 37.

[0434] Implementation Scheme 39: An RNA encoded by any one of the polynucleotide sequences described in Implementation Schemes 31 to 37.

[0435] Implementation Scheme 40: A vector comprising the polynucleotide described in any one of Implementation Schemes 31 to 39.

[0436] Implementation Scheme 41: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0437] Implementation Scheme 42: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141, and optionally comprising a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947.

[0438] Implementation Scheme 43: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising any one of the amino acids shown in SEQ ID NO: 165-860, and optionally comprising a polynucleotide containing any one of the posttranscriptional regulatory elements shown in SEQ ID NO: 945-947.

[0439] Implementation Scheme 44: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an amino acid represented by any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element represented by any one of SEQ ID NO: 945-947.

[0440] Implementation Scheme 45: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide containing the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0441] Implementation Scheme 46: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide containing a polynucleotide sequence shown in any one of SEQ ID NO: 925-944, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0442] Implementation Scheme 47: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0443] Implementation Scheme 48: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141, and optionally comprising a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947.

[0444] Implementation Scheme 49: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising any one of the amino acids shown in SEQ ID NO: 165-860, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0445] Implementation Scheme 50: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an amino acid represented by any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element represented by any one of SEQ ID NO: 945-947.

[0446] Implementation Scheme 51: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide containing the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0447] Implementation Scheme 52: A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide containing a polynucleotide sequence shown in any one of SEQ ID NO: 925-944, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

[0448] Implementation Scheme 53: The carrier as described in any of Implementation Schemes 40 to 52, wherein the carrier is an expression carrier.

[0449] Implementation Scheme 54: A vector as described in any of Implementation Schemes 40 to 52, wherein the vector is a transfer plasmid or a viral vector.

[0450] Implementation Scheme 55: A vector as described in any of Implementation Schemes 40 to 52, wherein the vector is a plasmid.

[0451] Implementation Scheme 56: A vector as described in any of Implementation Schemes 40 to 54, wherein the vector is a viral vector selected from adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, parvovirus vectors, rhabdovirus vectors, vesicular virus vectors, paramyxovirus vectors, measles virus vectors, hennipa virus vectors, alphavirus vectors, flavivirus vectors, retroviral vectors, and lentiviral vectors (LVV).

[0452] Implementation Scheme 57: The vector as described in Implementation Scheme 56, wherein the lentiviral vector is genome-engineered or derived from a lentivirus selected from: HIV (HIV type 1 or HIV type 2); Vesner-Medy virus (VMV); Capillary Arthritis-Encephalitis Virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV).

[0453] Implementation Scheme 58: A lentiviral vector comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi (Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding or containing a promoter operatively linked to the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and a poly(A) tail.

[0454] Implementation Scheme 59: A lentiviral vector comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding or containing a promoter operatively linked to a polynucleotide sequence shown in any one of SEQ ID NO: 925-944; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and a poly(A) tail.

[0455] Implementation Scheme 60: An RNA comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding a promoter operatively linked to the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and an optional poly(A) tail.

[0456] Implementation Scheme 61: An RNA comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding a promoter operatively linked to a polynucleotide sequence shown in any one of SEQ ID NO: 925-944; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and an optional poly(A) tail.

[0457] Implementation Scheme 62: A lentiviral vector as described in Implementation Scheme 58 or 59, wherein the vector encodes the promoter sequence shown in SEQ ID NO: 949 or 950.

[0458] Implementation Scheme 63: RNA as described in Implementation Scheme 60 or 61, wherein the RNA encodes the promoter sequence shown in SEQ ID NO: 949 or 950.

[0459] Implementation Scheme 64: A recombinant lentivirus comprising one or more copies of a lentiviral vector of any one of Implementation Schemes 58-60 or RNA of any one of Implementation Schemes 61-63.

[0460] Implementation Scheme 65: A composition comprising an antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes 1 to 5, a bispecific antibody as described in any one of Implementation Schemes 6 or 7, an antibody conjugate as described in any one of Implementation Schemes 8 to 10, a CAR as described in any one of Implementation Schemes 11 to 30, a polynucleotide as described in any one of Implementation Schemes 31 to 39, a vector as described in any one of Implementation Schemes 40 to 60, an RNA as described in any one of Implementation Schemes 61 to 63, or a recombinant lentivirus as described in Implementation Scheme 64.

[0461] All publications, patent applications and granted patents cited in this specification are incorporated herein by reference as if each individual publication, patent application or granted patent were specifically and individually incorporated by reference.

[0462] While the above embodiments have been described in detail through illustration and examples for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings considered herein, that certain changes and modifications may be made to the above embodiments without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

[0463] K. Example

[0464] Example 1

[0465] Recombinant Lentiviral

[0466] Delivery of functional anti-BCMA CAR to T cells

[0467] Recombinant T-cell-specific lentiviral particles were produced, which have a viral envelope expressing mutated viral envelope glycoproteins (fusion factors) and non-viral membrane-binding tropism molecules, and carry a lentiviral vector encoding anti-BCMA CAR. Figure 1 .

[0468] HEK293T cells were transfected with the following plasmids: a plasmid encoding a nonviral membrane-binding tropism molecule comprising an anti-CD3 scFv fused to the CD8α hinge and transmembrane domain; a plasmid encoding a mutant VSIV-G fusion factor comprising K47Q and R354A amino acid substitutions; a plasmid encoding lentivirus GAG / POL; a plasmid encoding lentivirus REV; and a transfer plasmid encoding a lentiviral vector comprising an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE element operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR.

[0469] Table 10 lists the reference numbers of recombinant lentiviruses (LV) and the corresponding SEQ ID NOs and CAR structures of the anti-BCMA CAR amino acid sequences.

[0470] Table 10

[0471] Jurkat functional titer

[0472] 1 x 10 5 Jurkat cells were seeded in each well of a 96-well plate. Cells were transduced using recombinant lentiviruses carrying a novel anti-BCMA CAR (LV1 to LV18) and a control anti-BCMA CAR obtained from the literature (LV19). Seven days after transduction, Jurkat cells were harvested, stained with recombinant phycoerythrin (PE)-labeled BCMA extracellular domain-FC fusion protein (BCMA-PE), and analyzed by flow cytometry. Functional titers, expressed as transduction units (TUs) per milliliter, were determined by measuring the number of transduced Jurkat cells. Figure 2A .

[0473] Anti-BCMA CAR expression

[0474] 5 x 10 5 Personal PBMCs were seeded in each well of a 24-well plate. Cells were transduced with recombinant lentivirus LV1 to LV19 at an MOI of 2 based on Jurkat functional titer, or in 0.5 mL volumes if MOI 2 was not feasible. Seven days post-transduction, PBMCs were harvested, stained with BCMA-PE, and analyzed by flow cytometry to assess the percentage of cells expressing anti-BCMA CAR. Figure 2B .

[0475] Anti-BCMA CAR activity

[0476] 5 x 10 4PBMCs transduced with recombinant lentivirus LV1 to LV19 and 5 x 10 4 5 x 10 BCMA-expressing tumor cells (RPMI-8226) or 5 x 10 4 Low BCMA-expressing tumor cells (Daudi) were co-cultured for 24 hours. Anti-BCMA CAR activity was assessed by collecting the co-culture supernatant and measuring IFNγ levels using the Meso Scale Discovery (MSD®) assay. The percentage of anti-BCMA CAR-positive cells was plotted against the IFNγ levels produced in the co-culture. Figure 2D .

[0477] Summarize

[0478] These data indicate that recombinant T cell-specific lentiviral particles (LV1 to LV18) carrying anti-BCMA CAR can transduce CD3-expressing cells, anti-BCMA CAR is expressed on PBMCs transduced with LV1 to LV18, and the anti-BCMA CAR expressed on transduced PBMCs can recognize cells with high or low BCMA expression and generate IFNγ in response to binding antigen.

[0479] Example 2

[0480] Lentiviral vector structure and expression and function of anti-BCMA CAR

[0481] Recombinant T-cell-specific lentiviral particles having a viral envelope expressing a mutated viral envelope glycoprotein (fusion factor) and a non-viral membrane-binding kinetic molecule; and carrying a lentiviral vector encoding various promoters, anti-BCMA CAR, no post-transcriptional response element (PRE), or with wild-type WPRE or mutated WPRE.

[0482] HEK293T cells were transfected with the following plasmids: a plasmid encoding a nonviral membrane-binding tropism molecule comprising an anti-CD3 scFv fused to the CD8α hinge and transmembrane domain; a plasmid encoding a mutant VSIV-G fusion factor comprising K47Q and R354A amino acid substitutions; a plasmid encoding lentivirus GAG / POL; a plasmid encoding lentivirus REV; and a transfer plasmid encoding a lentiviral vector comprising an MNDU3 promoter (SEQ ID NO: 950), an SFFV promoter (SEQ ID NO: 952), or an EF1α promoter (SEQ ID NO: 949) operably linked to a polynucleotide encoding a CD8α signal peptide and anti-BCMACAR, without a post-transcriptional response element, or having a wild-type WPRE (SEQ ID NO: 945) or a mutant WPRE (SEQ ID NO: 946) operably linked to the 3' end of a polynucleotide encoding anti-BCMACAR.

[0483] Table 11 lists the reference numbers of recombinant lentiviruses, the corresponding SEQ ID NOs of the amino acid sequences against BCMA CAR, and the structures of different lentiviral vectors.

[0484] Table 11

[0485] Infection titer

[0486] 1 x 10 5 Jurkat cells were seeded in each well of a 96-well plate and transduced with recombinant lentiviruses (including LV 19) listed in Table 11, said LV 19 containing a lentiviral vector encoding a control anti-BCMA CAR obtained from the literature. Cells were passaged three days after transduction. Cells were harvested seven days after transduction. Genomic DNA was isolated and purified from the harvested cells and used for quantitative PCR (qPCR) assays to determine the vector copy number (VCN), and subsequently to determine IU / mL. Figure 3A .

[0487] All lentiviral vector structures tested produced infectious titers and were subsequently used to transduce PBMCs.

[0488] VCN and anti-BCMA CAR expression

[0489] 5 x 10 5 Individual PBMCs were laid in each well of a 24-well plate and transduced with recombinant lentiviruses of volume-matched values ​​listed in Table 11.

[0490] Four days after transduction, PBMCs were passaged into 24-well GREX plates. Seven days after transduction, PBMCs were harvested, one cell sample was stained with BCMA-PE and analyzed by flow cytometry to assess the percentage of cells expressing anti-BCMA CAR, and another cell sample was used to isolate and purify genomic DNA for quantitative PCR (qPCR) to determine the vector copy number (VCN). Figure 3B .

[0491] These data indicate that different lentiviral vector structures combined with different anti-BCMA CAR combinations resulted in a range of transduction and anti-BCMA CAR expression.

[0492] Anti-BCMA CAR activity

[0493] 5 x 10 5 Personal PBMCs were seeded in each well of a 24-well plate and transduced using recombinant lentiviruses listed in Table 11, which had the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE. Except for LV 3.6, LV 3.8, LV 9.8, and LV 13.8, PBMCs were transduced at 1 MOI (based on IU / mL determined in Jurkat cells), with volume-matched lentiviruses used for LV 3.6, LV 3.8, LV 9.8, and LV 13.8. Four days after transduction, PBMCs were passaged into 24-well GREX plates. Seven days after transduction, PBMCs were harvested; one set of cells was stained with BCMA-PE and analyzed by flow cytometry to assess the number of anti-BCMA CAR-expressing cells, and the other set was used for co-culture assays to assess anti-BCMA CAR function.

[0494] 5 x 10 4 PBMCs with 5 x 10 transductions 4 RPMI-8226 cells were co-cultured for 24 hours. Anti-BCMACAR activity was assessed by harvesting the supernatant from the PBMC / RPMI-8226 cell co-culture and measuring IFNγ and IL-2 levels using MSD assay. The percentage of IFNγ and IL-2 levels generated during co-culture was plotted relative to the percentage of anti-BCMACAR positive cells. Figure 3C and 3D .

[0495] By culturing 5 x 10 cells without target cells 4PBMCs transduced for 24 hours were used to assess antigen-independent anti-BCMA CAR activity. After 24 hours, the supernatant was harvested, and IFNγ levels were measured using the MSD assay. IFNγ levels were plotted against the lentiviral structure used to express the anti-BCMA CAR. Figure 3E .

[0496] These data indicate that different combinations of lentiviral structures and anti-BCMA CAR can be selected to modulate the expression and activity of anti-BCMA CAR. Furthermore, the data showed that PBMCs expressing the anti-BCMA CAR shown in SEQ ID NO: 259, 263, 266, 270, 273, and 277 exhibited comparable or increased cell expansion and comparable or increased activity compared to the control anti-BCMA CAR, and only three combinations showed high levels of antigen-independent (persistent) signaling.

[0497] Off-target transduction

[0498] Off-target transduction of multiple myeloma cells was evaluated in two BCMA-expressing multiple myeloma cell lines, RPMI-8226 and KMS-11. 1 x 10 5 RPMI-8226 cells or 1 x 10 5 KMS-11 cells were seeded in each well of a 96-well plate and treated with recombinant lentiviruses listed in Table 11 at an MOI of 1. These lentiviruses had the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, EF1α promoter with no WPRE; LV 19; and LV 20. LV 20 is a recombinant lentiviral particle comprising a viral envelope expressing a nonviral membrane-binding tropism molecule comprising an anti-CD3 scFv fused to the CD8α hinge and transmembrane domain; a mutant VSIV-G fusion factor comprising K47Q and R354A amino acid substitutions; and a lentiviral vector comprising an MNDU3 promoter (SEQ ID NO: 950) operatively linked to a polynucleotide encoding a CD8α signal peptide and GFP, and a wild-type WPRE (SEQ ID NO: 945) operatively linked to the 3' end of a polynucleotide encoding GFP.

[0499] Three days after treatment, cells were passaged. Seven days after treatment, cells were harvested, genomic DNA was isolated and purified, and qPCR was performed to determine vector integration using VCN. The VCN value of anti-BCMA CAR was normalized to the VCN value of LV20, which expresses GFP instead of anti-BCMA CAR.

[0500] Data showed that the differences in off-target transduction in multiple myeloma were primarily driven by the specific anti-BCMA CAR expressed, rather than by any particular lentiviral vector structure. Several structures used to express anti-BCMA CAR in LV3, LV5, LV6, LV8, and LV9 showed low levels of off-target transduction, comparable to or lower than LV19 expressing the control anti-BCMA CAR. In contrast, LV13 exhibited the highest off-target transduction rate compared to the other LVs. Figure 3F .

[0501] Example 3

[0502] Lentiviral drugs administered in vivo showed antitumor efficacy in a mouse model of multiple myeloma.

[0503] The antitumor efficacy of in vivo administration of recombinant lentiviral particles was investigated in a mouse model of multiple myeloma. The recombinant lentiviral particles comprise an envelope expressing an anti-CD3 kinetic molecule and a mutant VSIV-G fusion factor, as well as a lentiviral vector encoding an anti-BCMA CAR.

[0504] The recombinant lentivirus for in vivo administration is produced by transient transfection of HEK293T cells with a plasmid encoding a nonviral membrane-binding tropism molecule containing an anti-CD3 scFv fused to the CD8α hinge and transmembrane domain; a mutant VSIV-G fusion factor containing K47Q and R354A amino acid substitutions; lentivirus GAG / POL; lentivirus REV; and a transfer plasmid encoding a lentiviral vector comprising: (i) an MNDU3 promoter (SEQ ID NO: 950) operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a wild-type WPRE (SEQ ID NO: 945) operably linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR; (ii) the MNDU3 promoter (SEQ ID NO: 950) operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a mutant WPRE (SEQ ID NO: 946) operably linked to an anti-BCMA CAR polynucleotide. (iii) The 3' end of the polynucleotide encoding the CAR; or (iv) the SFFV promoter (SEQ ID NO: 952) operatively linked to the polynucleotide encoding the CD8α signal peptide and the anti-BCMA CAR, and the mutated WPRE (SEQ ID NO: 946) operatively linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR; or (iv) the EF1α promoter (SEQ ID NO: 949) operatively linked to the polynucleotide encoding the CD8α signal peptide and the anti-BCMA CAR, but without PRE.

[0505] In this embodiment, the recombinant lentivirus reference number, the SEQ ID NO of the anti-BCMA CAR peptide, and the corresponding lentivirus structure shown in Table 12 were used.

[0506] Table 12

[0507] In vitro anti-BCMA CAR T cells were also prepared. Briefly, HEK293T cells were transiently transfected with the following plasmids: a plasmid encoding the wild-type VSIV-G fusion factor; a plasmid encoding the lentivirus GAG / POL; a plasmid encoding the lentivirus REV; and a transfer plasmid encoding a lentiviral vector containing an MNDU3 promoter operably linked to a CD8α signal peptide and a control anti-BCMA CAR (SEQ ID NO: 954) obtained from the literature, and a wild-type WPRE (SEQ ID NO: 945) operably linked to the 3' end of a polynucleotide encoding anti-BCMA CAR. PBMCs were then transduced with recombinant lentivirus and cultured for 7 days to generate anti-BCMA CAR T cells.

[0508] First Daudi Model Research

[0509] Administer 2 x 10⁻⁶ intravenously to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1 x 10⁻⁶ Daudi cells were administered intravenously to four of the five groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); or 2.2 x 10 8 IU at LV 3.1, LV 6.1, LV 8.1, or LV 13.1. Administer 5 x 10 to mice that have not received PBMC. 6 10 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0510] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo LV anti-BCMA CAR cells experienced tumor regression. Figure 4A .

[0511] Second Daudi Model Research

[0512] Administer 2 x 10⁻⁶ intravenously to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1 x 10⁸ Daudi cells were administered intravenously to eight of the nine groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 8 IU's LV 3.1, LV 6.1, LV 6.3, LV 8.1, LV 9.3, LV 9.6 or LV 13.8; or 5.6 x 10 7 IU LV 6.8. Administer 5 x 10⁻⁶ IU to mice not treated with PBMCs. 6 10 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0513] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo with some LV anti-BCMA CARs experienced mild control of tumor growth, while mice treated with LV 6.8 and LV 13.8 experienced durable tumor regression. Figure 4B .

[0514] Third Daudi Model Research

[0515] Administer 2 x 10⁻⁶ intravenously to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1 x 10⁸ Daudi cells were administered intravenously to eight of the nine groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 8 IU's LV 3.3, LV 3.6, LV 8.3, LV 8.6, LV 8.8, LV 13.3 or LV 13.6; or 5.6 x 10 7 IU LV 6.8. Administer 5 x 10⁻⁶ IU to mice not treated with PBMCs. 6 10 isolated anti-BCMA CART cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CART at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0516] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo with some LV anti-BCMA CARs experienced mild control of tumor growth, while mice treated with LV 6.8 and LV 8.8 experienced durable tumor regression. Figure 4C .

[0517] First RPMI Model Research

[0518] NOD scid-gamma (NSG) mice were subcutaneously injected with 1 x 10 6 One RPMI-8226 cell line (BCMA-positive tumor cell line). Tumor growth was allowed to reach approximately 110 mm. 3 Up to 140mm 3 The size (about two and a half weeks).

[0519] Then, five of the six groups of mice were administered 1x10 intravenously. 6 Human PBMC. The next day, give the recipient 1 x 10 6Mouse control with individual PBMCs administered the drug (DMEM); 5.0 x 10 7 IU at LV 6.3, LV 6.8, LV 8.3, or LV 8.8. Administer 2 x 10 IU to group 6 mice. 6 Unmodified in vitro anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured externally using calipers, and mice were euthanized at predetermined humanitarian endpoints based on tumor size and physical condition.

[0520] Tumor size increased in mice treated with the mediator control. Mice treated with LV 6.3 experienced moderate tumor regression, while mice treated with ex vivo anti-BCMA CAR T cells or in vivo with LV 6.8, LV 8.3, or LV 8.8 experienced complete and durable tumor regression. Figure 4D .

[0521] Administer 5 x 10 PBMCs to mice that have not received PBMCs. 6 10 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2.

[0522] Second RPMI Model Research

[0523] NOD scid-gamma (NSG) mice were subcutaneously injected with 1 x 10 6 One RPMI-8226 cell line (BCMA-positive tumor cell line). Tumor growth was allowed to reach approximately 110 mm. 3 Up to 140mm 3 The size (about two and a half weeks).

[0524] Then, 1 x 10⁻⁶ mice were administered intravenously to four of the five groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 7 IU's LV 6.8, 5.0 x 10 7 IU's LV 6.8 or 1.25 x 10 8 IU LV 6.8. Administer 2 x 10⁻⁶ IU to group 5 mice. 6 Unmodified in vitro anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration.5 IU recombinant human IL-2. Tumor volume was measured externally using calipers, and mice were euthanized at predetermined humanitarian endpoints based on tumor size and physical condition.

[0525] Mice treated with the vector control showed increased tumor size. Mice treated with all three doses of LV 6.8 experienced dose-dependent but complete and durable tumor regression. Mice treated with ex vivo anti-BCMA CAR T cells also experienced complete and durable tumor regression. Figure 4E .

[0526] Third RPMI Model Research

[0527] NOD scid-gamma (NSG) mice were subcutaneously injected with 1 x 10 6 One RPMI-8226 cell line (BCMA-positive tumor cell line). Tumor growth was allowed to reach approximately 110 mm. 3 Up to 140mm 3 The size (about two and a half weeks).

[0528] Then, three of the four groups of mice were administered 1 x 10 intravenously. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 5.0 x 10 7 IU's LV 6.3 or 1.25 x 10 8 IU LV 6.3. Administer 2 x 10⁻⁶ IU to the fourth group of mice. 6 Unmodified in vitro anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured externally using calipers, and mice were euthanized at predetermined humanitarian endpoints based on tumor size and physical condition.

[0529] Mice treated with the vector showed increased tumor size. Mice treated with two doses of LV 6.3 experienced dose-dependent tumor regression. Mice treated with ex vivo anti-BCMA CAR T cells experienced complete and durable tumor regression. Figure 4F .

[0530] Fourth Daudi model study

[0531] Administer 2 x 10⁻⁶ intravenously to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1 x 10⁻⁶ Daudi cells were administered intravenously to four of the five groups of mice.6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 8 IU's LV 6.1 or LV 6.3; or 5.6 x 10 7 IU LV 6.8. Administer 5 x 10⁻⁶ IU to mice not treated with PBMCs. 6 10 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 x 10⁻⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0532] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo with LV 6.1 and LV 6.3 experienced mild control of tumor growth, while mice treated with LV 6.8 experienced complete and durable tumor regression. Figure 4G .

[0533] Example 4

[0534] Comparison of antitumor efficacy in mouse models of multiple myeloma in vivo and in vitro

[0535] The antitumor efficacy of recombinant lentiviral particles was investigated in a mouse model of multiple myeloma. These particles comprised an envelope expressing an anti-CD3 kinetic molecule and a mutant VSIV-G fusion factor, as well as lentiviral vectors encoding various anti-BCMA CARs. The recombinant lentiviruses were formulated into lentiviral particles for in vivo administration and used to generate in vitro anti-BCMA CAR T cells.

[0536] Recombinant lentivirus was generated by transiently transfecting HEK293T cells with the following plasmids: a plasmid encoding a nonviral membrane-binding tropism molecule comprising anti-CD3scFv fused to the CD8α hinge and transmembrane domain; a plasmid encoding a mutant VSIV-G fusion factor comprising K47Q and R354A amino acid substitutions; a plasmid encoding lentivirus GAG / POL; a plasmid encoding lentivirus REV; and a transfer plasmid encoding a lentiviral vector encoding the anti-BCMA CAR or GFP control shown in SEQ ID NO: 429, SEQ ID NO: 954, or SEQ ID NO: 955.

[0537] Table 13

[0538] In this embodiment, the recombinant lentivirus reference number, the SEQ ID NO of the anti-BCMA CAR peptide, and the corresponding lentivirus structure shown in Table 12 were used.

[0539] Table 14

[0540] In vitro anti-BCMA CAR T cells were also prepared by transducing PBMCs with recombinant lentivirus and culturing the transduced cells for 7 days to generate anti-BCMA CAR T cells.

[0541] In vivo Daudi model study

[0542] Administer 2 x 10⁻⁶ intravenously to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1 x 10⁻⁶ Daudi cells were administered intravenously to four of the five groups of mice. 6 Individual PBMCs. The next day, mice that did not receive PBMCs were given a mediator control (DMEM), while mice that received PBMCs were given 5.0 x 10⁻⁶ ppm of the drug. 7 IU of LV 6.8, LV A, LV B, or LV 19 (GFP control). Tumor volume was measured using a bioluminescence imaging system.

[0543] Tumor size increased in mice treated with the vector, mice treated with a GFP control, and mice treated with a lentivirus expressing an anti-BCMA CAR containing the binding domains used in idecabtagenevicleucel. Mice treated with a lentivirus expressing an anti-BCMA CAR containing those binding domains as used in ciltacabtagene autoleucel experienced inhibition of tumor growth. Only mice treated with an anti-BCMA CAR containing SEQ ID NO: 429 experienced tumor regression. Figure 5A .

[0544] In vitro Daudi model study

[0545] Administer 2 x 10⁻⁶ intravenously to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Five days later, three of the five groups of mice were intravenously administered 2 x 10⁻⁶ Daudi cells. 6 Personal anti-BCMA CAR T cells. Mice that did not receive anti-BCMA CAR T cells were administered a mediator control (DMEM) or 2 x 10⁻⁶ cells. 6 10 untransduced control human T cells (UTD), while mice receiving anti-BCMACAR T cells were administered 2 x 10 6Anti-BCMA CAR T cells expressing CAR encoded by SEQ ID NO: 429, SEQ ID NO: 954, or SEQ ID NO: 955. Tumor volume was measured using a bioluminescence imaging system.

[0546] In mice treated with the vector and untransduced control T cells, tumor size increased. Mice treated with CAR T cells expressing an anti-BCMA CAR containing the binding domain used in idecabtagene vicleucel showed a transient reduction in tumor burden, while mice treated with CAR T cells expressing an anti-BCMA CAR containing SEQ ID NO: 429 or an anti-BCMA CAR similar to the binding domain used in ciltacabtagene autoleucel showed considerable and complete tumor regression. Figure 5B .

[0547] Generally, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and claims, but should be interpreted as including all possible embodiments together with equivalents to the full scope defined by the claims. Therefore, the claims are not limited by this disclosure.

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (a) A heavy chain variable region (VH), comprising CDRH1, CDRH2, and CDRH3 of the antibody or its antigen-binding fragment as shown in Table 1; a peptide linker; and a light chain variable region (VL), comprising CDRL1, CDRL2, and CDRL3 of the antibody or its antigen-binding fragment as shown in Table 1; or (b) VHH domains of CDRH1, CDRH2 and CDRH3 containing the antibodies or their antigen-binding fragments shown in Table 1.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein: (a) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 12, 13 and 14, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 16, 17 and 18; (b) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 22, 23 and 24, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 26, 27 and 28; (c) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 32, 33 and 34, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 36, 37 and 38; (d) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 42, 43 and 44, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 46, 47 and 48; (e) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 52, 53 and 54, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 56, 57 and 58; (f) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 62, 63 and 64, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 66, 67 and 68; (g) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 72, 73 and 74, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 76, 77 and 78; (h) VH comprises CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 82, 83 and 84, and VL comprises CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 86, 87 and 88; (i) VH contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 92, 93 and 94, and VL contains CDRL1, CDRL2 and CDRL3 containing the amino acid sequences shown in SEQ ID NO: 96, 97 and 98; (j) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 102, 103 and 104; (k) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 106, 107 and 108; (l) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 110, 111 and 112; (m) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 114, 115 and 116; (n) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 118, 119 and 120; (o) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 122, 123 and 124; (p) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 126, 127 and 128; (q) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 130, 131 and 132; (r) The VHH domain contains CDRH1, CDRH2 and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 134, 135 and 136; (s) The VHH domain contains CDRH1, CDRH2, and CDRH3 containing the amino acid sequences shown in SEQ ID NO: 138, 139, and 140; or (t) The VHH domain contains CDRH1, CDRH2, and CDRH3, which contain the amino acid sequences shown in SEQ ID NO: 142, 143, and 144.

3. The antibody or antigen-binding fragment thereof as described in claim 1 or claim 2, wherein: (a) VH contains the amino acid sequence shown in SEQ ID NO: 11, and VL contains the amino acid sequence shown in SEQ ID NO: 15; (b) VH contains the amino acid sequence shown in SEQ ID NO: 21, and VL contains the amino acid sequence shown in SEQ ID NO: 25; (c) VH contains the amino acid sequence shown in SEQ ID NO: 31, and VL contains the amino acid sequence shown in SEQ ID NO: 35; (d) VH contains the amino acid sequence shown in SEQ ID NO: 41, and VL contains the amino acid sequence shown in SEQ ID NO: 45; (e) VH contains the amino acid sequence shown in SEQ ID NO: 51, and VL contains the amino acid sequence shown in SEQ ID NO: 55; (f) VH contains the amino acid sequence shown in SEQ ID NO: 61, and VL contains the amino acid sequence shown in SEQ ID NO: 65; (g) VH contains the amino acid sequence shown in SEQ ID NO: 71, and VL contains the amino acid sequence shown in SEQ ID NO: 75; (h) VH contains the amino acid sequence shown in SEQ ID NO: 81, and VL contains the amino acid sequence shown in SEQ ID NO: 85; (i) VH contains the amino acid sequence shown in SEQ ID NO: 91, and VL contains the amino acid sequence shown in SEQ ID NO: 95; or (a) The VHH domain contains any one of the amino acid sequences shown in SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, wherein the polypeptide linker is selected from: TGEKP (SEQ ID NO: 2); (GGGGS) n , where n = 1, 2, 3, 4 or 5 (SEQ ID NO: 3, 976-979); EGKSSGSGSESKVD (SEQ ID NO: 4); KESGSVSSEQLAQFRSLD (SEQ ID NO: 5); LRQRDGERP (SEQ ID NO: 6); LRQKDGGGSERP (SEQ ID NO: 7); LRQKD(GGGS)2ERP (SEQ ID NO: 8), GETSTGSGGSGGSGGAD (SEQ ID NO: 9) and GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 10) and variants thereof containing amino acid sequences that are 95% identical to their amino acid sequences.

5. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

6. A bispecific antibody comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5.

7. The bispecific antibody of claim 6, wherein the bispecific antibody further comprises an anti-CD3 antibody that binds to CD3δ, CD3ε, CD3γ or CD3ζ.

8. An antibody conjugate comprising the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5.

9. The antibody conjugate of claim 8, wherein the antigen or its antigen-binding fragment is conjugated to a cytotoxic agent.

10. The antibody conjugate of claim 8 or claim 9, wherein: (a) The cytotoxic agent is selected from the following toxins: saponins, diphtheria toxin, Pseudomonas exotoxin A, ricin A chain derivatives, small molecule toxins and combinations thereof; (b) The cytotoxic agent is a radioactive isotope selected from the following: 131I, 90Y, 177Lu, 188Re, 67Cu, 213Bi, 211At and 227Ac; (c) The cytotoxic agent is selected from the following RNA polymerase II inhibitors and / or RNA polymerase III inhibitors: amatoxins, α-amaminine, β-amaminine, γ-amaminine, ε-amaminine, amatoxins, amatoxin amide, amatoxin non-toxic cyclic peptide, amatoxin acid, and any functional fragments, derivatives, or analogs thereof; or (d) The cytotoxic agent is selected from the following DNA damaging agents: anti-tubulin agents, DNA cross-linking agents, DNA alkylating agents and mitotic disruptors.

11. A chimeric antigen receptor (CAR), said chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5; a spacer domain; a transmembrane domain and one or more intracellular signal transduction domains.

12. The CAR of claim 11, wherein the spacer domain comprises a hinge domain selected from the following: CD4 hinge, CD8β hinge, CD8α hinge, CD28 hinge, CD134 hinge, CD137 hinge, CD152 hinge, CD278 hinge, IgG1 hinge, IgG2 hinge, IgG3 hinge, and IgG4 hinge.

13. The CAR of claim 11 or claim 12, wherein the spacer domain comprises an amino acid sequence shown in any one of SEQ ID NO: 145, 146, 147, 148, 149 and 150 or an amino acid sequence that is at least 95% identical thereto.

14. The CAR of any one of claims 11 to 13, wherein the transmembrane domain is isolated from or derived from a polypeptide selected from: α, β, γ or δ chains of T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amembrane protein (AMN), and programmed cell death 1 (PDCD1).

15. The CAR of any one of claims 11 to 14, wherein the transmembrane domain comprises an amino acid sequence shown in any one of SEQ ID NO: 151, 152, 153, 154, 155, 156 and 157 or an amino acid sequence that is at least 95% identical thereto.

16. The CAR of any one of claims 11 to 15, wherein the one or more intracellular signal transduction domains comprise major signal transduction domains isolated from or derived from a polypeptide selected from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

17. The CAR of any one of claims 11 to 16, wherein the one or more intracellular signal transduction domains comprise a major signal transduction domain isolated from CD3ζ.

18. The CAR of claim 17, wherein the primary signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical thereto.

19. The CAR of any one of claims 11 to 18, wherein the one or more intracellular signal transduction domains comprise co-stimulatory signal transduction domains isolated from or derived from a polypeptide selected from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, ICAM, CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70.

20. The CAR of any one of claims 11 to 19, wherein the one or more intracellular signal transduction domains comprise a co-stimulatory signal transduction domain, the co-stimulatory signal transduction domain comprising an amino acid sequence shown in any one of SEQ ID NO: 159, 160, 161, 162, 163 and 164 or an amino acid sequence that is at least 95% identical thereto.

21. A CAR comprising: an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, 90, 101 or 117; a spacer domain comprising an amino acid sequence shown in any one of SEQ ID NO: 145, 146 and 148, or an amino acid sequence at least 95% identical thereto; a transmembrane domain comprising an amino acid sequence shown in SEQ ID NO: 151 or 153; one or more intracellular signal transduction domains comprising a co-stimulatory signal transduction domain comprising an amino acid sequence shown in any one of SEQ ID NO: 159, 160 and 162, or an amino acid sequence at least 95% identical thereto; and further comprising a major signal transduction domain comprising an amino acid sequence shown in SEQ ID NO: 158, or an amino acid sequence at least 95% identical thereto.

22. A CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 165-860.

23. A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282 and 283.

24. A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379 and 380.

25. A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 and 452.

26. A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547 and 548.

27. A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619 and 620.

28. A CAR comprising the amino acid sequence shown in any one of SEQ ID NO: 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715 and 716.

29. The CAR of any one of claims 11 to 28, further comprising a signal peptide.

30. The CAR of claim 29, wherein the signal peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872 and 873.

31. A polynucleotide encoding a CAR, comprising the polynucleotide sequence shown in any one of SEQ ID NO: 905-924.

32. A polynucleotide encoding a signal peptide and a CAR, comprising the polynucleotide sequence shown in any one of SEQ ID NO: 925-944.

33. A polynucleotide encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, a bispecific antibody as described in claim 6 or 7, an antibody conjugate as described in any one of claims 8 to 10, or a CAR as described in any one of claims 11 to 30.

34. A polynucleotide encoding or comprising a promoter operatively linked to any one of claims 31 to 33.

35. The polynucleotide of claim 34, wherein the promoter comprises the polynucleotide sequence shown in any one of SEQ ID NO: 980, 981, 982, 983, 984 and 985.

36. The polynucleotide of claim 34 or claim 35, further comprising a post-transcriptional response element.

37. The polynucleotide of claim 36, wherein the post-transcriptional response element comprises the polynucleotide sequence shown in any one of SEQ ID NO: 945, 946 and 947.

38. A DNA comprising the polynucleotide sequence of any one of claims 31 to 37.

39. An RNA encoded by any one of claims 31 to 37.

40. A vector comprising the polynucleotide sequence of any one of claims 31 to 39.

41. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 11-144, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

42. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141, and optionally comprising a polynucleotide containing any one of the post-transcriptional regulatory elements shown in SEQ ID NO: 945-947.

43. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising any one of the amino acids shown in SEQ ID NO: 165-860, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

44. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an amino acid represented by any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element represented by any one of SEQ ID NO: 945-947.

45. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide containing the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

46. ​​A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 950, the promoter being operatively linked to a polynucleotide containing a polynucleotide sequence shown in any one of SEQ ID NO: 925-944, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

47. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 11-144, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

48. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof containing any one of the amino acids shown in SEQ ID NO: 20, 30, 39, 50, 59, 70, 80, 90, 100, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

49. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising any one of the amino acids shown in SEQ ID NO: 165-860, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

50. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide encoding a signal peptide and a chimeric antigen receptor, the chimeric antigen receptor comprising an amino acid represented by any one of SEQ ID NO: 189, 237, 261, 333, 357, 429, 477, 525, 573, 597, 621, 645, 669, 693, 717, 741, 765, 789, 813, and 837, and optionally comprising a polynucleotide containing a post-transcriptional regulatory element represented by any one of SEQ ID NO: 945-947.

51. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide containing the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

52. A vector encoding or comprising a promoter containing the sequence shown in SEQ ID NO: 949, the promoter being operatively linked to a polynucleotide containing a polynucleotide sequence shown in any one of SEQ ID NO: 925-944, and optionally containing a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 945-947.

53. The vector as claimed in any one of claims 40 to 52, wherein the vector is an expression vector.

54. The vector according to any one of claims 40 to 52, wherein the vector is a transfer plasmid or a viral vector.

55. The vector according to any one of claims 40 to 52, wherein the vector is a plasmid.

56. The vector according to any one of claims 40 to 54, wherein the vector is a viral vector selected from adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, parvovirus vectors, rhabdovirus vectors, vesicular virus vectors, paramyxovirus vectors, measles virus vectors, hennipa virus vectors, alphavirus vectors, flavivirus vectors, retrovirus vectors, and lentivirus vectors (LVV).

57. The vector of claim 56, wherein the lentiviral vector is genome-engineered or derived from a lentivirus selected from: HIV (HIV type 1 or HIV type 2); Vesner-Medy virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV).

58. A lentiviral vector comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi (Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding or containing a promoter operatively linked to the polynucleotide sequence shown in SEQ ID NO: 904 and any one of the polynucleotide sequences shown in SEQ ID NO: 905-924; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and a poly(A) tail.

59. A lentiviral vector comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi(ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding or containing a promoter operatively linked to a polynucleotide sequence shown in any one of SEQ ID NO: 925-944; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and a poly(A) tail.

60. The lentiviral vector of claim 58 or claim 59, wherein the lentiviral vector encodes a promoter comprising the polynucleotide sequence shown in SEQ ID NO: 949 or 950.

61. An RNA comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi (Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding a promoter operatively linked to a polynucleotide sequence shown in SEQ ID NO: 904 and any one of SEQ ID NO: 905-924; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and an optional poly(A) tail.

62. An RNA comprising: a 5' long terminal repeat (LTR) containing an R region and a U5 region; a Psi (Ψ) packaging signal; cPPT / FLAP; an output element; a polynucleotide encoding a promoter operatively linked to a polynucleotide sequence shown in any one of SEQ ID NO: 925-944; an optional WPRE; a 3' LTR containing a U3 region and an R region; a polyadenylation signal; and an optional poly(A) tail.

63. The RNA of claim 61 or claim 62, wherein the lentiviral vector encodes a promoter comprising the polynucleotide sequence shown in SEQ ID NO: 949 or 950.

64. A recombinant lentivirus comprising one or more copies of the lentiviral vector of any one of claims 58-60 or the RNA of any one of claims 61-63.

65. A composition comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, a bispecific antibody as claimed in claim 6 or 7, an antibody conjugate as claimed in any one of claims 8 to 10, a CAR as claimed in any one of claims 11 to 30, a polynucleotide as claimed in any one of claims 31 to 39, a vector as claimed in any one of claims 40 to 60, RNA as claimed in any one of claims 61 to 63, or a recombinant lentivirus as claimed in claim 64.

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