CN118369332A - CD 33-targeting antigen recognizing receptor and use thereof - Google Patents

CD 33-targeting antigen recognizing receptor and use thereof Download PDF

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CN118369332A
CN118369332A CN202280071579.3A CN202280071579A CN118369332A CN 118369332 A CN118369332 A CN 118369332A CN 202280071579 A CN202280071579 A CN 202280071579A CN 118369332 A CN118369332 A CN 118369332A
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amino acid
acid sequence
set forth
sequence set
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A·丹尼安
R·J·布伦特延斯
I·C·洛伦兹
A·卡恩
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Cancer And Related Diseases Memorial Hospital
Sloan Caitlin Cancer Research Association
Three Institution Treatment Discovery Institute Inc
Memorial Sloan Kettering Cancer Center
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Cancer And Related Diseases Memorial Hospital
Sloan Caitlin Cancer Research Association
Three Institution Treatment Discovery Institute Inc
Memorial Sloan Kettering Cancer Center
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Priority claimed from PCT/US2022/042444 external-priority patent/WO2023034560A1/en
Publication of CN118369332A publication Critical patent/CN118369332A/en
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Abstract

The presently disclosed subject matter provides antigen recognizing receptors specifically targeting CD33 and cells comprising such CD 33-targeting antigen recognizing receptors. The presently disclosed subject matter further provides for the use of the CD 33-targeting antigen recognizing receptor for therapy.

Description

CD 33-targeting antigen recognizing receptor and use thereof
Cross Reference to Related Applications
The present application claims priority from U.S. provisional patent application No. 63/306,395, filed 2 nd 2022, and U.S. provisional patent application No. 63/240,196, filed 9 nd 2021, the contents of which are incorporated by reference in their entirety and claim priority from this provisional patent application.
Sequence listing
The present application contains a sequence listing that has been submitted via the EFS-Web and is hereby incorporated by reference in its entirety. The sequence listing created at month 31 of 2022 is named 0727341387.Xml and is 164,036 bytes in size.
1. Technical field
The presently disclosed subject matter provides methods and compositions for immunotherapy. The presently disclosed subject matter relates to antigen recognizing receptors (e.g., chimeric Antigen Receptors (CARs)) that specifically target CD33, cells comprising such receptors, and methods of treatment using such cells.
2. Background art
Acute Myeloid Leukemia (AML) is the most common and deadliest acute leukemia in the united states. The standard of care has been chemotherapy in the last forty years, with the recent addition of CD33 antibody-drug conjugates (ADCs) and targeting small molecules in medical devices. Despite these new supplements, AML remains a devastating disease with a 5-year survival of less than 30%. Thus, new AML interventions are urgently needed.
In the last decade, CAR T cells have become one of the most effective forms of immunotherapy. Although many communities have used this technology for the treatment of AML via targeting CD33, there is still a major obstacle: the density of such antigens is non-uniform within and between patient populations. This is very important because CAR T cell efficacy has been demonstrated to correlate with antigen density, and a reduction in antigen surface expression, rather than complete loss, is the mechanism of CAR T cell immune escape. To overcome this problem, membrane proximal epitope targeting has become a strategy to enhance CAR T cell efficacy, thereby enabling an increase in the efficacy of whole CAR T cells and the ability of effector cells to recognize low antigen density variants. Thus, developing CAR T cells targeting CD33 membrane proximal epitopes would allow for enhanced elimination of diseases with different CD33 expression, including splice variants that may lack membrane distal domains, which are targets for all currently known antibodies.
3. Summary of the invention
The presently disclosed subject matter provides antigen recognizing receptors specifically targeting CD33 and cells comprising such CD 33-targeting antigen recognizing receptors. The presently disclosed subject matter further provides for the use of the CD 33-targeting antigen recognizing receptor for therapy.
The presently disclosed subject matter provides an antigen recognizing receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain specifically binds to CD 33. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv). In certain embodiments, the variable region is positioned from N-terminus to C-terminus: v H-VL. In certain embodiments, the extracellular antigen-binding domain is a human scFv. In certain embodiments, the extracellular antigen-binding domain is a Fab, which is optionally crosslinked. In certain embodiments, the extracellular antigen-binding domain is F (ab) 2. In certain embodiments, one or more of scFv, fab, and F (ab) 2 are included in a fusion protein having a heterologous sequence to form an extracellular antigen-binding domain.
In certain embodiments, the heavy chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 14 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 23 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 24 or a conservative modification thereof;
(d) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 33 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 34 or a conservative modification thereof;
(e) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39 or a conservative modification thereof;
(f) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 48 or a conservative modification thereof;
(g) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 58 or a conservative modification thereof;
(h) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 67 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68 or a conservative modification thereof;
(i) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 77 or a conservative modification thereof; or alternatively
(J) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87 or a conservative modification thereof.
In certain embodiments, the heavy chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 14 or a conservative modification thereof; or alternatively
(C) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 23 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 24 or a conservative modification thereof.
In certain embodiments, the light chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising SEQ ID No. 17 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising SEQ ID No. 27 or a conservative modification thereof;
(d) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 40 or a conservative modification thereof, and a CDR2 comprising SEQ ID NO. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 42 or a conservative modification thereof;
(e) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51 or a conservative modification thereof;
(f) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61 or a conservative modification thereof;
(g) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71 or a conservative modification thereof;
(h) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80 or a conservative modification thereof; or alternatively
(I) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 42 or a conservative modification thereof.
In certain embodiments, the light chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising SEQ ID No. 17 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof.
In some embodiments of the present invention, in some embodiments,
(A) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 5, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 6, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 7;
(b) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 15, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 16, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 17;
(c) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 25, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 26, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 27;
(d) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 32, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 33, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 34; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 25, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 26, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 27;
(e) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 39; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 40, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 41, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 42;
(f) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 47, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 48; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 49, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 50, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 51;
(g) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 56, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 58; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 59, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 60, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 61;
(h) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 66, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 67, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 68; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 69, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 70, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 71;
(i) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 76, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 77; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO:78, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:79, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 80; or alternatively
(J) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 85, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 86, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 87; and the light chain variable region comprises: CDR1, which CDR1 comprises the amino acid sequence shown in SEQ ID NO. 5, CDR2, which CDR2 comprises the amino acid sequence shown in SEQ ID NO. 41, and CDR3, which CDR3 comprises the amino acid sequence shown in SEQ ID NO. 42.
In some embodiments of the present invention, in some embodiments,
(A) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 5, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 6, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 7;
(b) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO.12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO.13, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; and the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO.15, a CDR2 comprising the amino acid sequence shown in SEQ ID NO.16, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 17; or alternatively
(C) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; and the light chain variable region comprises: CDR1, which CDR1 comprises the amino acid sequence shown in SEQ ID NO:25, CDR2, which CDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and CDR3, which CDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.
In certain embodiments, the heavy chain variable region comprises an amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an amino acid sequence set forth below. In certain embodiments, the heavy chain variable region comprises amino acid sequence ：SEQ IDNO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 as set forth below. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 8, SEQ ID NO. 18 or SEQ ID NO. 28.
In certain embodiments, the light chain variable region comprises an amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO 92 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an amino acid sequence set forth below. In certain embodiments, the light chain variable region comprises amino acid sequence ：SEQ IDNO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO. 92 as set forth below. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO.9, SEQ ID NO. 19 or SEQ ID NO. 29.
In certain embodiments, the heavy chain variable region of (a) comprises an amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous to the amino acid sequences shown below; and (b) the light chain variable region comprises an amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO 92 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the amino acid sequences shown below.
In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 set forth below; and (b) the light chain variable region comprises amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ IDNO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO. 92 as set forth below. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 8, SEQ ID NO. 18 or SEQ ID NO. 28; and (b) the light chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 9, SEQ ID NO. 19 or SEQ ID NO. 29.
In some embodiments of the present invention, in some embodiments,
(A) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9;
(b) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 19;
(c) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29;
(d) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 35 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29;
(e) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 44;
(f) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 52 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 53;
(g) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 63;
(h) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 73;
(i) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 81, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 82;
(j) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 89; or alternatively
(K) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 93.
In some embodiments of the present invention, in some embodiments,
(A) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9;
(b) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 19; or alternatively
(C) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 29.
In certain embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99 or SEQ ID NO 100. In certain embodiments, the signal peptide is covalently attached to the 5' end of the extracellular antigen-binding domain.
In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, or a combination thereof.
In certain embodiments, the intracellular signaling domain comprises a cd3ζ polypeptide. In certain embodiments, the intracellular signaling domain further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.
In certain embodiments, the antigen recognizing receptor is a Chimeric Antigen Receptor (CAR) or a T cell-like fusion protein. In certain embodiments, the antigen recognizing receptor is a CAR.
In certain embodiments, the antigen recognizing receptor is recombinantly expressed. In certain embodiments, the antigen recognizing receptor is expressed by a vector. In certain embodiments, the vector is a gamma retroviral vector.
The presently disclosed subject matter provides cells comprising the presently disclosed antigen recognizing receptors. In certain embodiments, the cells are transduced with antigen recognizing receptors. In certain embodiments, the antigen recognizing receptor is constitutively expressed on the surface of the cell.
In certain embodiments, the cell is an immune response cell. In certain embodiments, the cell is a cell of a stranguria line or a cell of a myeloid line. In certain embodiments, the cell is selected from the group consisting of: t cells, natural Killer (NK) cells, and stem cells from which lymphoid cells may be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a Cytotoxic T Lymphocyte (CTL) or regulatory T cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
The presently disclosed subject matter further provides nucleic acids encoding the presently disclosed antigen recognizing receptors. The presently disclosed subject matter further provides vectors comprising the presently disclosed nucleic acid molecules. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a gamma retroviral vector.
In addition, the presently disclosed subject matter provides host cells that express the nucleic acid molecules disclosed herein. In certain embodiments, the host cell is a T cell.
The presently disclosed subject matter further provides compositions comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
The presently disclosed subject matter also provides lipid nanoparticles comprising the nucleic acids disclosed herein. The presently disclosed subject matter also provides compositions comprising the lipid nanoparticles disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
The presently disclosed subject matter further provides methods of treating or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering the presently disclosed cells or compositions to a subject.
The presently disclosed subject matter also provides methods of reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject the presently disclosed cells or compositions. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and/or eradicates the tumor in the subject.
The presently disclosed subject matter provides methods of treating and/or preventing a tumor in a subject. In certain embodiments, the method comprises administering to the subject the presently disclosed cells or compositions. The presently disclosed subject matter provides methods of increasing or extending survival of a subject having a tumor. In certain embodiments, the method comprises administering to the subject the presently disclosed cells or compositions. In certain embodiments, the method reduces or eradicates tumor burden in the subject.
In certain embodiments, the disease or condition is a tumor. In certain embodiments, the tumor is a hematological cancer or a solid tissue cancer. In certain embodiments, the tumor is selected from the group consisting of: acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), myeloproliferative neoplasms (MPN), and chronic myelogenous neoplasms. In certain embodiments, the tumor is Acute Myeloid Leukemia (AML). In certain embodiments, the subject is a human.
The presently disclosed subject matter further provides a kit for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and/or preventing a tumor in a subject, and/or increasing or extending survival of a subject having a tumor, the kit comprising a presently disclosed cell, nucleic acid, or composition. In certain embodiments, the kit further comprises written instructions for using the presently disclosed cells or compositions to treat or ameliorate a disease or disorder in a subject, reduce tumor burden in a subject, treat and/or prevent a tumor in a subject, and/or increase or extend survival of a subject with a tumor.
In addition, the presently disclosed subject matter provides methods of producing a CD 33-targeted antigen recognizing receptor comprising introducing into a cell a nucleic acid encoding the antigen recognizing receptor.
Finally, the presently disclosed subject matter provides the cells or compositions disclosed herein for treating or ameliorating a disease or disorder in a subject. In certain embodiments, the disease or condition is a tumor. In certain embodiments, the tumor is a cancer. In certain embodiments, the disease or condition is selected from the group consisting of: lung neuroendocrine tumors, extrapulmonary neuroendocrine cancers, melanoma, neuroendocrine prostate cancer, breast cancer, gastrointestinal neuroendocrine tumors, pancreatic cancer, medullary thyroid cancer, small cell bladder cancer, ovarian small cell cancer, low grade glioma, glioblastoma, and neuroblastoma. In certain embodiments, the lung neuroendocrine tumor is selected from the group consisting of: lung neuroendocrine cancer, large cell neuroendocrine cancer, and small cell lung cancer. In certain embodiments, the tumor is small cell lung cancer. In certain embodiments, the subject is a human
4. Description of the drawings
The following detailed description, given by way of example and not intended to limit the invention to the specific embodiments described, may be understood with reference to the accompanying drawings.
Fig. 1A and 1B depict CD33 domain architecture. Fig. 1A shows full length CD33 (CD 33M) with two Ig-like domains in the extracellular region (IgV and IgC 2), a single transmembrane segment, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in the cytoplasmic region. In the common CD33 variant (CD 33 m), igV domains are deleted due to alternative splicing. FIG. 1B depicts CD33 gene structure with and without alternate splicing. Removal of exon 2 results in loss of IgV domain. A variety of antibodies that have entered the clinic in various forms (CAR, biTE, radiation therapy) target IgV domains. "GO" represents gemtuzumab ozogamicin.
Fig. 2 depicts an exemplary CD 33-targeting CAR structure according to the presently disclosed subject matter. CD 33-targeting CARs comprise truncated EGFR (EGFRt), an anti-CD 33 scFv, a Myc tag, a transmembrane domain comprising a CD28 polypeptide, and an intracellular signaling domain comprising a CD3 ζ polypeptide, and a costimulatory signaling region comprising the CD28 polypeptide. T2A is the cleavage site.
Figure 3 depicts surface expression levels and flow cytometry analysis of transduced PBMCs, demonstrating transduction efficiency and CAR surface expression of each construct detected by anti-Myc tag antibodies.
Figures 4A and 4B depict cytotoxicity of membrane proximal CARs. Fig. 4A shows the heavy and light chain variable region tests 3P14 and 4B2 and H195 reference CARs for each antibody oriented in V H-VL (HL) or V L-VH (LH) on the U937 cell line. Figure 4B shows testing of 3P14 and 4B2 and H195 reference CARs on OCi-AML3 cell lines.
Fig. 5A and 5B depict cytotoxicity of a membrane distal CAR. Figure 5A shows in vitro killing of U937 cells by CAR T cells generated from 5 antibodies targeting a membrane distal epitope in the CD33 IgV domain using cells from donor 2. Figure 5B shows in vitro killing of U937 cells by CAR T cells generated from 5 antibodies targeting a membrane distal epitope in the CD33 IgV domain using cells from donor 3.
Figures 6A through 6F depict the in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007 CAR. Figures 6A to 6C show cytotoxicity of 3 AML target lines containing GFP-firefly luciferase reporter gene (gL) of donor 1 at increased indicated effector to target ratios. Figures 6D to 6F show cytotoxicity of 3 AML target lines containing GFP-firefly luciferase reporter gene (gL) of donor 2 at increased indicated effector to target ratios. Killing was assessed using a luciferase assay. Efficient killing of TDI-Y-006 and TDI-Y-007 was observed in various CD33+ target cells and PBMC from different donors.
Figures 7A to 7D depict cytotoxicity of 1j19 CAR. FIG. 7A shows cytotoxicity as a percent of killing of a GFP-firefly luciferase reporter (gL) -containing HL60 cell line of donor 2 at an increased indicated effector to target ratio. FIG. 7B shows cytotoxicity as a percent of killing of the GFP-firefly luciferase reporter (gL) -containing HL60 cell line of donor 3 at an increased indicated effector to target ratio. FIG. 7C shows cytotoxicity as a percent of killing of the U937 cell line containing GFP-firefly luciferase reporter (gL) of donor 2 at increased indicated effector to target ratios. FIG. 7D shows cytotoxicity as a percent of killing of donor 1, an OCI-AML3 cell line containing the GFP-firefly luciferase reporter (gL) at an increased indicated effector to target ratio. Killing was assessed using a luciferase assay. Overall, an effective killing of 1J19 was observed compared to the H195 reference CAR.
Figures 8A to 8D depict CD33 CAR T cell proliferation after binding to target cells. Figures 8A and 8B show co-culture of human T cells transduced with lead CAR with U937 cells (CD 33 high) from two donors. Figures 8C and 8D show co-culture of human T cells transduced with a lead CAR with OCi-AML3 (CD 33 low) cells from two donors. The total number of remaining target cells and T cells was detected using flow cytometry 7, 14 and 21 days after co-cultivation. Total remaining human T cells per mL over time (in days) are shown.
Figures 9A to 9D depict cytokine secretion profiles of lead CD33 CAR T cells. Groups of 4 cytokines were analyzed for three different donors. FIG. 9A shows the measurement of GMCSF levels in supernatants collected after 24 hours of co-culture of target cells and effector cells using a human 12-fold Luminex panel kit. FIG. 9B shows the levels of INF-gamma. FIG. 9C shows TNF- α levels. FIG. 9D shows IL-2 levels. Data from representative donor 1 is shown.
Figures 10A and 10B depict the in vivo efficacy of lead CAR T in a U937 AML xenograft mouse model. Figure 10A shows imaging and quantification of U937 cells (CD 33 high) labeled with GFP-firefly luciferase over time in NCG mice treated with lead CAR T cell IV. Fig. 10B shows a survival curve.
Fig. 11A and 11B depict the in vivo efficacy of titrated administered lead CAR T. Figure 11A shows data for animals injected with 5.10 5 CAR T cells and monitored for tumor growth and survival. Figure 11B shows data for animals injected with 2.5.10 5 CAR T cells.
Figures 12A and 12B depict in vivo efficacy of lead CAR T in a OCi-AML3 xenograft mouse model. Imaging and quantification of OCi-AML3 cells (CD 33 low) labeled with firefly luciferase over time in NCG mice treated with lead CAR T cell IV. Figure 12A shows the average survival of 5 animals per group. Figure 12B shows individual survival curves for each animal.
Figure 13 depicts a direct comparison of the in vivo efficacy of two lead CARs and one spare CAR in a U937 AML xenograft mouse model. In NCG mice treated with lead and backup 1j19 CAR T cell IV, U937 cells labeled with firefly luciferase (CD 33 high) were imaged and quantified over time.
Figure 14 depicts the in vivo efficacy of TDI-Y-006CAR T on patient-derived AML xenografts. Patient-derived tumor cells were infused intravenously and allowed to grow for 10 days. TDI-Y-006CAR T was injected on day 14. Seven days later, the number of CD2-cd33+ cells was determined by flow cytometry. Mean data for H195 jdel from five mice and TDI-Y-006CAR T from four mice are shown, with p values < 0.05.
Fig. 15A and 15B depict pro-inflammatory cytokine levels in the PDX model. FIG. 15A shows IFN-gamma levels measured from TDI-Y-006CAR T treated mice using the human 12-fold Luminex panel kit after patient-derived xenograft implantation. FIG. 15B shows TNF- α levels. * p value <0.05.
Figures 16A and 16B depict in vitro assessment of toxicity of CD33 CAR T. Figure 16A shows hematopoietic stem cells isolated from umbilical cord with and without CD33 pilot CAR T tested by colony forming unit. FIG. 16B shows the total number of colonies used to assess toxicity of the experimental agents. As controls, gemtuzumab ozogamicin (GO, anti-CD 33 ADC) and Del CAR were used.
FIG. 17 depicts a heat map showing the relative binding of selected antibodies to tumor lines expressing human full length CD33, CD33-IgC and U937 CD 33.
FIGS. 18A to 18D depict the selection of scFv targeting membrane proximal CD 33-IgC. Figure 18A shows quantification of hybridoma-derived monoclonal antibody cross-reactivity as measured by His-tag binding. FIG. 18B shows quantification of binding kinetics of hybridoma-derived monoclonal antibodies to CD33 in solution. Figure 18C shows quantification of EC50 of leader hybridoma-derived monoclonal antibodies to overexpressed (3T 3) and AML cell line (U937). FIG. 18D shows epitope binning quantification of selected hybridoma-derived monoclonal antibodies.
Fig. 19 depicts a representative flow cytometry plot demonstrating comparable retroviral transduction efficiencies determined by flow cytometry. P-values were determined by repeated measures of one-way ANOVA. Data are mean ± SEM of three independent experiments; ns, is not significant.
Figures 20A through 20G depict membrane proximal-targeted CAR T cells that enhance in vitro function and proliferation capacity. Fig. 20A shows flow cytometry histograms and quantitative geometric Mean Fluorescence (MFI), depicting the expression of fluorescence-labeled CD33 on wild-type or CD33 knockout (CD 33 KO) AML cells detected with anti-CD 33 antibodies. Fig. 20B shows a 24 hour D-luciferin assay, which demonstrates lysis of U937-CD33 high gfpLuc + tumor cells (n=4; * P <0.0001; * P <0.001; * P <0.05, according to two-way ANOVA). Data are mean ± SEM of four independent experiments. Figure 20C shows a 24 hour D-luciferin assay, which demonstrates the lysis of OCiAML-CD 33 low gfpLuc + tumor cells (n=4;, P <0.05, according to two-way ANOVA). Data are mean ± SEM of four independent experiments. Fig. 20D shows a 138 hour (6 days) assay, which demonstrates the lysis of U937-CD33 high gfpLuc + tumor cells at low effector to target ratio (n=2;, P <0.01;, P <0.05, according to two-way ANOVA). Data represent four independent experiments. Fig. 20E shows a 138 hour assay, which demonstrates the lysis of OCiAML3-CD33 low gfpLuc + tumor cells at low effector to target ratios (n=2; * P <0.01; * P <0.05, according to two-way ANOVA). Data represent four independent experiments. Figure 20F shows quantification of flow cytometry analysis demonstrating enhancement of CAR T cell proliferation targeting membrane proximal CD33 in the presence of U937-CD33 high tumor cells (n=9;, P <0.0001, according to two-factor ANOVA). Data are mean ± SEM of three independent experiments. Figure 20G shows quantification of flow cytometry analysis demonstrating enhancement of CAR T cell proliferation targeting membrane proximal CD33 in the presence of OCiAML-CD 33 low gfpLuc + tumor cells (n=9; * P <0.0001, according to two-way ANOVA). Data are mean ± SEM of three independent experiments; ns, is not significant.
Figures 21A to 21D depict the specificity of membrane proximal targeting of CAR T cells. FIG. 21A shows a flow cytometry histogram depicting FLAG expression on transduced U937-CD33IgCgfpLuc + tumor cells. FIG. 21B shows a 24 hour D-luciferin assay, which demonstrates the lysis of U937-CD33IgCgfpLuc + tumor cells. Data represent two independent experiments. FIG. 21C shows a 24 hour D-luciferin assay, which demonstrates the lysis of U937-CD33KOgfpLuc + tumor cells. Data represent two independent experiments. Fig. 21D shows quantification of total colonies generated using a Colony Forming Unit (CFU) assay to measure hematopoietic killing, where total colony count is the combination of BFU-E, GEMM-CFU and GM-CFU (n=9;, P <0.0001;, P <0.001, according to a common single factor ANOVA; ns, not significant). The data shown are the mean ± SEM combination of three independent experiments.
Fig. 22A-22G depict membrane proximal targeting CAR T cells featuring unique in vitro activation profiles. FIG. 22A shows the 24-hour cytokine secretion profile of Tc1/Th1 cytokines detected by human 12-fold Luminex panel when co-cultured with U937-CD33 high gfpLuc +, OCiAML-CD 33 low gfpLuc +、U937-CD33IgCgfpLuc+ tumor and U937-CD33KOgfpLuc + (n=3; * P <0.0001; * P <0.05, according to Dunnett multiple comparison test against H195DEL control). Data represent three independent experiments. Figure 22B shows quantification of flow cytometry analysis showing intracellular production of CAR T cells of Tc1/Th1 activating cytokines (n=4; P <0.0001;, P <0.01;, P <0.05, according to two-factor ANOVA; ns, not significant). Data are mean ± SEM of four independent experiments. FIG. 22C shows a qualitative representation of CD4 +CAR+ and CD8 +CAR+ Tc1/Th1 activating cytokine secretion. Figure 22D shows quantification of flow cytometry analysis showing CD4 + CAR T cell intracellular production of Tc1/Th1 activating cytokines (n=4;, P <0.0001,;, P <0.05, according to two-way ANOVA). Data are mean ± SEM of four independent experiments. Figure 22E shows quantification of flow cytometry analysis showing CD8 + CAR T cell intracellular production of Tc1/Th1 activating cytokines (n=4; * P <0.001; * P <0.01; * P <0.05, according to two-way ANOVA). Data are mean ± SEM of four independent experiments. Figure 22F shows quantification of flow cytometry analysis demonstrating CAR T cell activation status (n=4;, P <0.0001, according to two-factor ANOVA) 7 days after antigen stimulation. Data are mean ± SEM of three independent experiments. Figure 22G shows a qualitative representation of flow cytometry analysis comparing CAR T cell activation profiles. Data were pooled from three independent experiments.
Fig. 23A-23K depict membrane proximal CD 33-targeting CAR T cells that enhance survival in a xenograft mouse model. Fig. 23A shows a schematic of an in vivo experimental setup. NCG mice were vaccinated with U937-CD33 high gfpLuc + tumor, followed by treatment with CAR T cells. Figure 23B shows the survival of NCG mice bearing U937-CD33 high gfpLuc + tumors and treated with 5.0 x 10 5 CAR T cells (n=5; * P < 0.01). The P value for the survival period was determined by the log rank Mantel-Cox test, 95% confidence interval. In vivo data shown are pooled from 5 mice. Fig. 23C shows the survival of NCG mice bearing U937-CD33 high gfpLuc + tumors and treated with 2.5 x 10 5 CAR T cells (n=5; P <0.01; P < 0.05). The P value for the survival period was determined by the log rank Mantel-Cox test, 95% confidence interval. In vivo data shown are pooled from 5 mice. Figure 23D shows the survival of NCG mice bearing U937-CD33 high gfpLuc + tumors and treated with 1.0 x 10 5 CAR T cells (n=5;. P <0.01; ns, not significant). The P value for the survival period was determined by the log rank Mantel-Cox test, 95% confidence interval. Figure 23E shows the survival of NCG mice bearing U937-CD33 high gfpLuc + tumors and treated with 5.0 x 10 4 CAR T cells (n=5; * P < 0.05). The P value for the survival period was determined by the log rank Mantel-Cox test, 95% confidence interval. Figure 23F shows imaging of U937-CD33 height gfpLuc + over time in tumor-bearing NCG mice treated with 5.0 x 10 5 CAR T cells. Figure 23G shows tumor regression in NCG mice vaccinated with U937-CD33 high gfpLuc + tumors and subsequently treated with 5.0 x 10 5 CAR T cells. Fig. 23H shows a schematic of an in vivo experimental setup. NCG mice were vaccinated with OCiAML-CD 33 low gfpLuc + tumor followed by treatment with CAR T cells. Figure 23I shows the survival of NCG mice vaccinated with OCiAML-CD 33 low gfpLuc + tumor and treated with 5.0 x 10 5 CART cells (n=5; * P < 0.05). The P value for the survival period was determined by the log rank Mantel-Cox test, 95% confidence interval. Figure 23J shows imaging of OCiAML-CD 33 low gfpLuc + in tumor-bearing NCG mice treated with 5.0 x 10 5 CAR T cells over time. Figure 23K shows tumor regression in NCG mice carrying OCiAML3-CD33 low gfpLuc + tumors and treated with 5.0 x 10 5 CAR T cells.
Figures 24A to 24D depict membrane proximal CD33 targeting CAR T cells that reduce tumor burden in patient-derived AML xenograft models. Figure 24A shows flow cytometry histograms and geometric Median Fluorescence Intensity (MFI) of CD33 expression on AML60B patient samples detected with fluorescence labeled CD33 specific antibodies and isotype control. Fig. 24B shows a schematic of experimental setup of a patient-derived xenograft model. NCG mice were vaccinated with patient-derived AML blasts and treated with allogeneic CAR T cells. Bone marrow aspirate was analyzed 28 days after tumor inoculation. Figure 24C shows quantification of flow cytometry analysis demonstrating a reduction in tumor burden in mice treated with CAR T cells targeting membrane proximal CD33 (n=6;, P <0.05, according to common single factor ANOVA; ns, not significant). Fig. 24D shows survival of NCG mice bearing AML60B patient-derived tumors and treated with either membrane distal or membrane proximal CAR T cells (n=6; P <0.01, P < 0.05). The P value for the survival period was determined by the log rank Mantel-Cox test, 95% confidence interval. The data shown were pooled from two independent experiments using two different healthy donors.
5. Detailed description of the preferred embodiments
The presently disclosed subject matter provides antigen recognizing receptors (e.g., chimeric Antigen Receptors (CARs)) that specifically target CD 33. The presently disclosed subject matter further provides cells comprising such receptors. The cell may be an immune response cell, such as a genetically modified immune response cell (e.g., a T cell or a Natural Killer (NK) cell). The presently disclosed subject matter also provides methods of treatment using such cells, e.g., for treating and/or ameliorating a disease or disorder associated with CD33 (e.g., AML).
The specification and examples describe non-limiting embodiments of the present disclosure.
For the sake of clarity of the disclosure, and not by way of limitation, the detailed description is divided into the following subsections:
5.1. Definition;
5.2.CD33；
5.3. an antigen recognizing receptor;
5.4. a cell;
5.5. a composition and a carrier;
5.6. A polypeptide;
5.7. Formulations and administration;
5.8. A method of treatment;
5.9. a kit; and
5.10. Exemplary embodiments.
5.1. Definition of the definition
Unless defined otherwise, 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 belongs. The following references provide the skilled artisan with a general definition of many of the terms used in the present invention: singleton et al Dictionary of Microbiology and MolecularBiology (1994, 2 nd edition); the Cambridge Dictionary of SCIENCE AND Technology (Walker, 1988); the Glossary of Genetics, 5 th edition, R.Rieger et al (eds.), SPRINGER VERLAG (1991); and Hale & Marham, THE HARPER Collins Dictionary of Biology (1991). The following terms as used herein have the meanings given below, unless otherwise indicated.
As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 3 or more standard deviations according to practice in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times the value, and more preferably within 2 times.
By "immunoresponsive cell" is meant a cell or progenitor or progeny thereof that plays a role in the immune response. In certain embodiments, the immune response cell is a gonococcal cell. Non-limiting examples of lineage cells include T cells, natural Killer (NK) cells, B cells, and stem cells from which lymphoid cells may be differentiated. In certain embodiments, the immune response cell is a myeloid cell.
By "activating an immune response cell" is meant inducing a change in signal transduction or protein expression in the cell, resulting in the initiation of an immune response. For example, a signal transduction cascade is generated when the CD3 chain aggregates in response to ligand binding and immune receptor tyrosine-based inhibitory motifs (ITAMs). In certain embodiments, when an endogenous TCR or exogenous CAR binds to an antigen, formation of an immune synapse occurs, including a number of molecules that aggregate in proximity to the bound receptor (e.g., CD4 or CD8, CD3 gamma/delta/epsilon/zeta, etc.). This aggregation of membrane-bound signaling molecules causes the ITAM motif contained in the CD3 chain to be phosphorylated. This phosphorylation in turn initiates T cell activation pathways, ultimately activating transcription factors such as NF-. Kappa.B and AP-1. These transcription factors induce overall gene expression in T cells to increase IL-2 production for major regulation of proliferation and expression of T cell proteins, thereby initiating T cell mediated immune responses.
By "stimulating an immune response cell" is meant a signal that results in a robust and sustained immune response. In various embodiments, this occurs after immune cell (e.g., T cell) activation or concomitantly mediated by receptors including, but not limited to, CD28, CD137 (4-1 BB), OX40, CD40, and ICOS. Receiving multiple stimulation signals may be important to establish a robust and long-term T cell-mediated immune response. T cells may be rapidly inhibited and not responsive to antigens. While the effects of these costimulatory signals may vary, they generally result in increased gene expression to produce long-term survival, proliferation, and anti-apoptotic T cells that produce a robust response to antigen to achieve complete and sustained eradication.
As used herein, the term "antigen recognizing receptor" refers to a receptor capable of recognizing a target antigen (e.g., CD 33). In certain embodiments, the antigen recognition receptor is capable of activating an immune or immune response cell (e.g., T cell) upon binding to a target antigen.
As used herein, the term "antibody" means not only an intact antibody molecule, but also a fragment of an antibody molecule that retains the ability to bind an immunogen. Such fragments are also well known in the art and are commonly employed in vitro and in vivo. Thus, as used herein, the term "antibody" means not only an intact immunoglobulin molecule, but also the well-known active fragments F (ab') 2 and Fab. F (ab') 2 and Fab fragments lacking the Fe fragment of the intact antibody, cleared more rapidly from the circulation, and possibly have less non-specific tissue binding of the intact antibody (Wahl et al, nucleic Med (1983); 24:316-325). As used herein, comprises all natural antibodies, bispecific antibodies; a chimeric antibody; fab, fab', single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies. In certain embodiments, an "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H) and a heavy chain constant (C H) region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V L) and a light chain constant C L region. The light chain constant region is composed of one domain, C L. The V H region and V L region can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V H and V L consists of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) as well as the first component of the classical complement system (C1 q).
As used herein, a "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable region of an immunoglobulin heavy and light chain. See, for example, kabat et al Sequences ofProteins of Immunological Interest, 4 th edition U.S. Pat. No. HEALTH AND Human Services, national Institutes of Health (1987), or the IMGT numbering system (Lefranc, the Immunologist (1999); 7:132-136; lefranc et al, dev. Comp. Immunol. (2003); 27:55-77). Typically, an antibody comprises three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide most of the contact residues for binding of antibodies to antigens or epitopes. In certain embodiments, CDR regions are described using the IMGT numbering system. In some embodiments, CDR regions are delineated using the IMGT numbering system accessible at http:// www.imgt.org/IMGT_ vquest/input.
As used herein, the term "single chain variable fragment" or "scFv" is a fusion protein in which the variable regions of the heavy (V H) and light (V L) chains of an immunoglobulin (e.g., mouse or human) are covalently linked to form a V H: VL heterodimer. The heavy chain (V H) and the light chain (V L) are linked either directly or through a linker (e.g., 10, 15, 20, 25 amino acids) encoding a peptide that links the N-terminus of V H to the C-terminus of V L or the C-terminus of V H to the N-terminus of V L. Linkers are typically glycine-rich for flexibility, and serine or threonine-rich for solubility. The linker may connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al, anal. Chem.80 (6): 1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entirety. In certain embodiments, the linker is a G4S linker.
In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 95, which provides the following:
GGGGSGGGGSGGGSGGGGS[SEQ ID NO:95]
in certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 96, which provides the following:
GGGGSGGGGSGGGGS[SEQ ID NO:96]
In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 97, which provides the following:
GGGGSGGGGSGGGGSGGGSGGGGS[SEQ ID NO:97]
in certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 98, which provides the following:
GGGGSGGGGSGGGGSGGGGSGGGSGGGGS[SEQ ID NO:98]
In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO 99, which provides the following:
GGGGS[SEQ ID NO:99]
In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 100, which provides the following:
GGGGSGGGGS[SEQ ID NO:100]
The scFv proteins retain the original immunoglobulin specificity despite removal of the constant region and introduction of the linker. Single chain Fv polypeptide antibodies may be expressed from nucleic acids comprising the V H and V L coding sequences, such as Huston et al Proc.Nat. Acad.Sci.USA, (1988); 85:5879-5883; U.S. Pat. nos. 5,091,513, 5,132,405, and 4,956,778; as described in U.S. patent publication nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., zhao et al, hyrbidoma (Larchmt) (2008); 27 (6): 455-51; peter et al, JCachexia Sarcopenia Muscle (2012); 8 months 12 days; shieh et al, J Imunol (2009); 183 (4): 2277-85; giomarelli et al, thromb Haemost (2007); 97 (6): 955-63; fife et al, J CLIN INVST (2006); 116 (8): 2252-61; brocks et al, immunotechnology 1993 (3): 173-84; moosmyer et al, ther Immunol 1995 (10:31-40); 8); agonistic scFvs (Peter et al, J Biol chemn (2003); 25278 (38): 36740-7; xie et al, nat Biotech 1997 15 (8 768-71; ledbeteter et al, crrenol 1997 (17); 17-6); 55 (257-6); 1636.
As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen binding domain and a transmembrane domain, the extracellular antigen binding domain being fused to an intracellular signaling domain capable of activating or stimulating an immune responsive cell. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv may be derived from fusing the variable heavy and light chain regions of an antibody. Alternatively or additionally, the scFv may be derived from Fab' (rather than from an antibody, e.g. obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain, and then fused to an intracellular signaling domain. By "substantially identical" or "substantially homologous" is meant that the polypeptide or nucleic acid molecule exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleotide sequence (e.g., any of the nucleotide sequences described herein). In certain embodiments, such sequences are at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the sequence of the amino acid or nucleic acid for comparison.
Sequence identity may be achieved by using sequence analysis software (e.g., university of madison university in wisconsin, lane 1710 wisconsin university biotechnology center, the sequence analysis software package BLAST, BESTFIT, GAP or the PILEUP/PRETTYBOX program of the genetic computer group, postal code 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically comprise substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, wherein the probability score between e-3 and e-100 represents closely related sequences.
As used herein, the percent homology between two amino acid sequences corresponds to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology%o=the #/total #. Times.100 of positions of identical positions), which takes into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences may be accomplished using mathematical algorithms.
The percent homology between two amino acid sequences can be determined using the algorithm of E.Meyers and W.Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12 and gap penalty 4. In addition, the percent homology between two amino acid sequences can be determined using Needleman and Wunsch (j.mol. Biol.48:444-453 (1970)) algorithms (which have been incorporated into the GAP program in the GCG software package (available at www.gcg.com)) using the Blossom62 matrix or PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1,2,3, 4,5 or 6.
Additionally or alternatively, the amino acid sequence of the presently disclosed subject matter can be further used as a "query sequence" to search a public database, for example, to identify related sequences. Such a search can be performed using the XBLAST program of Altschul et al (1990) J.mol.biol.215:403-10 (version 2.0). BLAST protein searches can be performed using the XBLAST program with a score = 50 and a word length = 3 to obtain amino acid sequences homologous to the specific sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m 900). To obtain a gapped alignment for comparison purposes, gapped BLAST may be utilized, as described in the following: altschul et al, (1997) Nucleic Acids Res.25 (17): 3389-3402. When utilizing BLAST programs and gapped BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
An "effective amount" is an amount sufficient to affect a beneficial or desired clinical outcome upon treatment. An effective amount may be administered to a subject in one or more doses. In certain embodiments, an effective amount may be an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow the progression of the disease or otherwise reduce the pathological consequences of the disease. The effective amount may be determined by the physician on a case-by-case basis and is within the ability of one skilled in the art. In determining the appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells administered.
As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of a presently disclosed CD 33-targeting CAR (e.g., extracellular antigen binding domain) comprising an amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen binding domains of the presently disclosed CARs by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups based on their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid in the same group. For example, amino acids can be categorized by charge: positively charged amino acids include lysine, arginine, histidine; negatively charged amino acids include aspartic acid, glutamic acid; neutral charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In addition, amino acids can be categorized by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, tyrosine; the nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region may be replaced by other amino acid residues from the same group, and altered antibodies may be tested for retained function (i.e., the functions described in (c) through (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a particular sequence or CDR region are altered.
As used herein, the term "endogenous" refers to a nucleic acid molecule or polypeptide that is typically expressed in a cell or tissue.
As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" will encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides. "exogenous" nucleic acid means a nucleic acid that is not present in a native wild-type cell; for example, the exogenous nucleic acid may differ from the endogenous counterpart in sequence, position/location, or both. For clarity, the exogenous nucleic acid may have the same or different sequence relative to its natural endogenous counterpart; it may be genetically engineered into the cell itself or its progenitor cells, and may optionally be linked to alternative control sequences, such as non-native promoter or secretion sequences.
By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or sample obtained from a cell. The nucleic acid may be from another organism, or it may be an mRNA molecule that is not normally expressed, for example, in a cell or sample.
"Increasing" means positively changing by at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.
By "reduced" is meant a negative change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.
The term "isolated", "purified" or "biologically pure" refers to materials that are found in their natural state to varying degrees, often accompanied by components thereof. "separation" means the degree of separation from the original source or the surrounding environment. "purification" means a degree of separation that is greater than separation. A "purified" or "biologically pure" protein is sufficiently free of other materials that any impurities do not materially affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and uniformity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein substantially produces a band in the electrophoresis gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins, which can be purified separately.
By "isolated cell" is meant a cell that is separated from the molecules and/or cellular components that naturally accompany the cell.
As used herein, the term "antigen binding domain" refers to a domain capable of specifically binding to a particular epitope or group of epitopes present on a cell.
"Recognize" means to selectively bind to a target. T cells that recognize a tumor may express a receptor (e.g., CAR) that binds to a tumor antigen.
"Signal sequence" or "leader sequence" means a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs the protein into the secretory pathway.
By "specifically binds" or "specifically binds to" or "specifically targets" is meant a polypeptide or fragment thereof that recognizes and/or binds to a biomolecule of interest (e.g., a polypeptide, such as a CD33 polypeptide), but does not substantially recognize and/or bind to other molecules in a sample (e.g., a biological sample that naturally includes the presently disclosed polypeptide (e.g., a CD33 polypeptide)). In certain embodiments, the presently disclosed antigen recognizing receptors bind CD33 (e.g., human CD 33) with a dissociation constant (K D) of about 1x 10 -8 M or less, about 5x 10 -9 M or less, about 1x 10 -9 M or less, about 5x 10 -10 M or less, about 1x 10 -10 M or less, about 5x 10 -11 M or less, or about 1x 10 -11 M or less.
As used herein, the term "derivative" refers to a compound that is derived from some other compound and retains its general structure. For example, but not limited to, chloroform (chloroform) is a derivative of methane.
The terms "include," "comprising," and "includes" are intended to have the broad meaning given to them in the united states patent laws and may mean "including," "comprises," and the like.
As used herein, "treatment" refers to a clinical intervention that attempts to alter the disease process of the individual or cell being treated, and may be performed for prophylaxis or during the clinical pathology process. Therapeutic effects of treatment include, but are not limited to, preventing disease occurrence or recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or moderating the disease state, and alleviating or improving prognosis. By preventing the progression of the disease or disorder, the treatment may prevent exacerbations due to the disorder in the subject being affected or diagnosed or in the subject suspected of having the disorder, and the treatment may prevent the risk of having the disorder or the symptoms of the onset of the disorder or the disorder in the subject suspected of having the disorder.
An "individual" or "subject" herein is a vertebrate, such as a human or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sports animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; a rabbit; a dog; a cat; sheep; pig; a goat; cattle; a horse; and non-human primates, such as apes, monkeys.
Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the scope of the presently disclosed subject matter.
5.2.CD33
CD33 is a member of the single transmembrane molecule and sialic acid binding immunoglobulin (Ig) -like lectin (Siglec) family. CD33 consists of two extracellular domains IgV and IgC2 with immunoglobulin-like folds (see fig. 1A). CD33 has 3 subtypes produced via alternative splicing, with subtype 3 lacking the IgV domain (Ehninger et al, bloodCancer J, e218 (2014), sanford et al, leuk Lymphoma, 1965-1968 (2016), and Haubner et al, leukemia 33,64-74 (2019)). Recent studies have shown that about 50% of AML patients have CD33 Single Nucleotide Polymorphism (SNP) (rs 12459419C > T), resulting in expression of alternatively spliced CD33 subtype lacking exon 2, resulting in elimination of IgV domains (Bakker et al CANCER RES, 8443-8450 (2004)). In these patients, gemtuzumab Ozogamicin (GO), a CD 33-targeting antibody-drug conjugate (ADC), had no effect and increased risk of relapse, probably because the ADC failed to kill AML cells expressing this CD33 subtype lacking IgV. All currently available CD 33-targeting products necessarily suffer from this problem due to their epitopes in the IgV domain (see fig. 1B) (Perna et al, CANCER CELL, 506-519e505 (2017)).
In certain embodiments, the antigen recognizing receptor binds to human CD 33. In certain embodiments, human CD33 comprises or consists of an amino acid sequence having the following UniProt reference numbers: p20138-1 (SEQ ID NO: 1) or a fragment thereof. SEQ ID NO. 1 is provided below. In certain embodiments, CD33 comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In certain embodiments, the extracellular domain comprises or consists of amino acids 18 to 259 of: SEQ ID NO. 1. In certain embodiments, the transmembrane domain comprises or consists of amino acids 260 to 282 of: SEQ ID NO. 1. In certain embodiments, the cytoplasmic domain comprises or consists of amino acids 283 to 364: SEQ ID NO. 1.
MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYW
FREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRM
ERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWL
SAAPTSLGPRTTHSSVLI ITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTT
GIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTH
PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSE
VRTQ[SEQ ID NO:1]
In certain embodiments, CD33 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to the amino acid sequence set forth in seq id no: SEQ ID NO. 1.
In certain embodiments, the antigen recognizing receptor binds to a portion of human CD 33. In certain embodiments, the antigen recognizing receptor binds to the extracellular domain of CD 33. In certain embodiments, the extracellular domain of CD33 comprises an Ig-like V-type domain and an Ig-like C2-type domain. In certain embodiments, the extracellular domain of CD33 comprises an Ig-like C2 type. In certain embodiments, the Ig-like V-type domain comprises or consists of amino acids 19 to 135 of: SEQ ID NO. 1. In certain embodiments, the Ig-like C2 type domain comprises or consists of amino acids 145 to 228 of: SEQ ID NO. 1.
5.3. Antigen recognizing receptor
The presently disclosed antigen recognizing receptor specifically targets or binds to CD 33. In certain embodiments, the antigen recognizing receptor is a Chimeric Antigen Receptor (CAR). In certain embodiments, the antigen recognizing receptor is a TCR-like fusion molecule.
The presently disclosed subject matter also provides nucleic acid molecules encoding the presently disclosed antigen recognizing receptors. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide disclosed herein that targets the CD33 antigen recognition receptor.
5.3.1. Extracellular antigen binding domains
In certain embodiments, the extracellular antigen-binding domain of the antigen recognition receptor binds to CD 33.
In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a murine scFv. In certain embodiments, scfvs are identified by screening a scFv phage library with an antigen-Fc fusion protein.
In certain embodiments, the extracellular antigen-binding domain is a Fab. In certain embodiments, the Fab is crosslinked. In certain embodiments, the extracellular antigen-binding domain is F (ab) 2.
Any of the foregoing molecules may be included in a fusion protein having a heterologous sequence to form an extracellular antigen-binding domain. In certain non-limiting embodiments, the extracellular antigen binding domain (embodied as, for example, an scFv) binds to CD33 (e.g., human CD 33) with a binding affinity, for example, having the following dissociation constant (K D): 1X 10 -8 M or less, e.g., about 1X 10 -8 M or less, About 5 x 10 -9 M or less, about 1 x 10 -9 M or less, about 5 x 10 -10 M or less, about 1 x 10 -10 M or less, or about 1 x 10 -11 M or less. in certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of about 5 x 10 -9 M or less. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of about 5 x 10 -9 M. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of about 1 x 10 -9 M. in certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of between about 1 x 10 -9 M and about 5 x 10 -9 M. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of between about 1 x 10 -9 M and about 2x 10 -9 M. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of about 5 x 10 -9 M. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to CD33 (e.g., human CD33, e.g., soluble human CD 33) with a dissociation constant (K D) of about 1 x 10 -9 M.
In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) at a half maximal effective concentration (EC 50) value of about 1nM to about 50nM, about 5nM to about 50nM, about 10nM to about 50nM, about 20nM to about 50nM, about 30nM to about 50nM, about 40nM to about 50nM, or greater than about 50 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 1nM to about 5 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 2 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 2.16 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 5nM to about 10 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 10 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 8.5 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 40nM to about 50 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 45 nM. In certain embodiments, the presently disclosed extracellular antigen-binding domains bind to cells expressing CD33 (e.g., AML cells expressing CD 33) with an EC50 value of about 45 nM.
Binding of the extracellular antigen binding domain can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assay. Each of these assays typically detects the presence of a particular protein-antibody complex of interest by employing a labeling reagent (e.g., an antibody or scFv) that is specific for the complex of interest. For example, scFv can be radiolabeled and used in Radioimmunoassays (RIA) (see, e.g., month 3 of ,Weintraub,B.,Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques,The Endocrine Society,1986, incorporated herein by reference). The radioisotope may be detected by gamma or scintillation counting or by autoradiography or the like. In certain embodiments, the extracellular antigen-binding domain that targets CD33 is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include Green Fluorescent Protein (GFP), blue fluorescent protein (e.g., EBFP2, azurin, and mKalama a), cyan fluorescent protein (e.g., ECFP, azurin, and CyPet), and yellow fluorescent protein (e.g., YFP, lemon yellow, venus, and YPet). In certain embodiments, the CD 33-targeting human scFv is labeled with GFP.
In certain embodiments, CDRs are identified according to the IMGT numbering system.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No.3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof. SEQ ID NOS.2 to 4 are provided in Table 1.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v L comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof. SEQ ID NOS 5 to 7 are provided in Table 1.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No.5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 7.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 8. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to: SEQ ID NO. 8. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 8. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 8 is shown in SEQ ID NO. 10. SEQ ID NOS 8 and 10 are provided in Table 1 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 9. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to: SEQ ID NO. 9. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 9. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 9 is shown in SEQ ID NO. 11. SEQ ID NOS 9 and 11 are provided in Table 1 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO. 8, and V L, which comprises the amino acid sequence shown in SEQ ID NO. 9. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated as "3-P14". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 1
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 14 or a conservative modification thereof. SEQ ID NOS 12 to 14 are provided in Table 2.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v L comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 17 or a conservative modification thereof. SEQ ID NOS 15 to 17 are provided in Table 2.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 13 or a conservative modification thereof, a CDR3 comprising the amino acid sequence shown in SEQ ID No. 14 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 17 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO.12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:15, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 18. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 18. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 18. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 18 is shown in SEQ ID NO. 20. SEQ ID NOS 18 and 20 are provided in Table 2 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 19. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 19. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 19. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 19 is shown in SEQ ID NO. 21. SEQ ID NOS 19 and 21 are provided in Table 2 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:18, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 19. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "4-B2". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 2
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 23 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 24 or a conservative modification thereof. SEQ ID NOS.22 to 24 are provided in Table 3.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v L comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof. SEQ ID NOS 25 to 27 are provided in Table 3.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 23 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 24 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:25, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 28. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 28. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 28. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 28 is shown in SEQ ID NO. 30. SEQ ID NOS 28 and 30 are provided in Table 3 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 29. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 29. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 29. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 29 is shown in SEQ ID NO. 31. SEQ ID NOS 29 and 31 are provided in Table 3 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO. 28, and V L, which comprises the amino acid sequence shown in SEQ ID NO. 29. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-J19". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 3 Table 3
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 33 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 34 or a conservative modification thereof. SEQ ID NOS 32 to 34 are provided in Table 4.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v L comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof. SEQ ID NOS.25 to 27 are provided in Table 4.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 33 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 34 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 32, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 33, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 34; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:25, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 35. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 35. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 35. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 35 is shown in SEQ ID NO. 36. SEQ ID NOS.35 and 36 are provided in Table 4 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 29. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 29. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 29. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 29 is shown in SEQ ID NO. 31. SEQ ID NOS 29 and 31 are provided in Table 4 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:35, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 29. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-J19-2". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 4 Table 4
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39 or a conservative modification thereof. SEQ ID NOS: 37 to 39 are provided in Table 5.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v L comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 40 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 42 or a conservative modification thereof. SEQ ID NOS.40 to 42 are provided in Table 5.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 40 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 42 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 37, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 39; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:40, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:41, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 42.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 43. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 43. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 43. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 43 is shown in SEQ ID NO. 45. SEQ ID NOS 43 and 45 are provided in Table 5 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 44. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 44. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 44. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 44 is shown in SEQ ID NO. 46. SEQ ID NOS 44 and 46 are provided in Table 5 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO. 43, and V L, which comprises the amino acid sequence shown in SEQ ID NO. 44. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-P13". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 5
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 48 or a conservative modification thereof. SEQ ID NOS 38, 47 and 48 are provided in Table 6.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51 or a conservative modification thereof. SEQ ID NOS.49 to 51 are provided in Table 6.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 48 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 47, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 48; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:49, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:50, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 52. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 52. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 52. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 52 is shown in SEQ ID NO. 54. SEQ ID NOS 52 and 54 are provided in Table 6 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 53. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 53. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 53. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 53 is shown in SEQ ID NO. 55. SEQ ID NOS 53 and 55 are provided in Table 6 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:52, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 53. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-P23". In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 6
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 58 or a conservative modification thereof. SEQ ID NOS 56 to 58 are provided in Table 7.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61 or a conservative modification thereof. SEQ ID NOS 59 to 61 are provided in Table 7.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 58 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 56, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 58; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:59, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:60, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 61.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 62. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 62. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 62. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 62 is shown in SEQ ID NO. 64. SEQ ID NOS 62 and 64 are provided in Table 7 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 63. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 63. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 63. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 63 is shown in SEQ ID NO. 65. SEQ ID NOS 63 and 65 are provided in Table 7 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:62, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 63. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-A20". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 7
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 67 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68 or a conservative modification thereof. SEQ ID NOS 66 to 68 are provided in Table 8.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71 or a conservative modification thereof. SEQ ID NOS 69 to 71 are provided in Table 8.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 67 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 66, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 67, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 68; and V L, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO:69, CDR2 comprising the amino acid sequence shown in SEQ ID NO:70, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 71.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 72. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 72. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO:72. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 72 is shown in SEQ ID NO. 74. SEQ ID NOS 72 and 74 are provided in Table 8 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 73. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 73. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO:73. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 73 is shown in SEQ ID NO. 75. SEQ ID NOS 73 and 75 are provided in Table 8 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:72, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 73. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-N3". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 8
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 77 or a conservative modification thereof. SEQ ID NOS 57, 76 and 77 are provided in Table 9.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80 or a conservative modification thereof. SEQ ID NOS.78 to 80 are provided in Table 9.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 77 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 76, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 77; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:78, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:79, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 80.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 81. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 81. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 81. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 81 is shown in SEQ ID NO. 83. SEQ ID NOS 81 and 83 are provided in Table 9 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 82. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 82. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence set forth in SEQ ID NO. 82. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 82 is set forth in SEQ ID NO. 84. SEQ ID NOS 82 and 84 are provided in Table 9 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:81, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 82. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "1-H19". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 9
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87 or a conservative modification thereof. SEQ ID NOS 85 to 87 are provided in Table 10.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 42 or a conservative modification thereof. SEQ ID NOS 5, 41 and 42 are provided in Table 10.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence set forth in SEQ ID No. 85 or a conservative modification thereof, V H comprising CDR2 comprising the amino acid sequence set forth in SEQ ID No. 86 or a conservative modification thereof, V H comprising CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 42 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 85, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 86, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 87; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:41, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 42.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 88. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 88. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 88. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 88 is shown in SEQ ID NO. 90. SEQ ID NOS 88 and 90 are provided in Table 10 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 89. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 89. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO:89. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 89 is set forth in SEQ ID NO. 91. SEQ ID NOS 89 and 91 are provided in Table 10 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO:88, and V L, which comprises the amino acid sequence shown in SEQ ID NO: 89. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, scFv is designated as "2-F18". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
Table 10
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof. SEQ ID NOS.2 to 4 are provided in Table 11.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof. SEQ ID NOS.5 to 7 are provided in Table 11.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof; and V L, comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO.2, a CDR2 comprising the amino acid sequence shown in SEQ ID NO.3, a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4; and V L, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and CDR3, the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 7.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 8. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V H, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 8. In certain embodiments, the extracellular antigen-binding domain comprises: v H comprising the amino acid sequence shown in SEQ ID NO. 8. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 8 is set forth in SEQ ID NO. 93. SEQ ID NOS 8 and 93 are provided in Table 11 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 92. For example, an extracellular antigen-binding domain (e.g., scFv) comprises V L, which comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to an amino acid sequence shown in seq id no: SEQ ID NO. 92. In certain embodiments, the extracellular antigen-binding domain comprises: v L comprising the amino acid sequence shown in SEQ ID NO. 92. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 92 is shown in SEQ ID NO. 94. SEQ ID NOS 92 and 94 are provided in Table 11 below.
In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises: v H, which comprises the amino acid sequence shown in SEQ ID NO. 8, and V L, which comprises the amino acid sequence shown in SEQ ID NO. 92. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is designated as "4-P3". In certain embodiments, V H and V L are connected by a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO. 96.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
TABLE 11
The V H and/or V L amino acid sequences having at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., ,SEQ ID NO: 8、SEQ ID NO: 9、SEQ ID NO: 18、SEQ ID NO: 19、SEQ ID NO: 28、SEQ ID NO: 29、SEQ ID NO: 35、SEQ ID NO: 43、SEQ ID NO: 44、SEQID NO: 52、SEQ ID NO: 53、SEQ ID NO: 62、SEQ ID NO: 63、SEQ ID NO: 72、SEQ ID NO: 73、SEQ ID NO: 81、SEQ ID NO: 82、SEQ ID NO: 88、SEQ ID NO: 89 or SEQ ID NO: 92) may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the specified sequence, but retain the ability to bind CD 33.
In certain embodiments, substitutions, insertions and/or deletions are made in a specific sequence (e.g., ,SEQ ID NO: 8、SEQ ID NO: 9、SEQ ID NO:18、SEQ ID NO: 19、SEQ ID NO: 28、SEQ ID NO: 29、SEQ ID NO: 35、SEQ ID NO: 43、SEQ ID NO: 44、SEQ ID NO: 52、SEQ ID NO: 53、SEQ ID NO: 62、SEQ ID NO: 63、SEQID NO: 72、SEQ ID NO: 73、SEQ ID NO: 81、SEQ ID NO: 82、SEQ ID NO: 88、SEQ ID NO: 89 or SEQ ID NO: 92) for a total of 1 to 10 amino acids. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs of the extracellular antigen-binding domain (e.g., in the FR). In certain embodiments, the extracellular antigen-binding domain comprises a V H and/or V L sequence selected from the group consisting of SEQ ID NOs 8, 9, 18, 19, 28, 29, 35, 43, 44, 52, 53, 62, 63, 72, 73, 81, 82, 88, 89, or 92, including post-translational modifications of the sequence (SEQ ID NOs 8, 9, 18, 19, 28, 29, 35, 43, 44, 52, 53, 62, 63, 72, 73, 81, 82, 88, 89, or 92).
In certain embodiments, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) cross-competes with a reference antibody or antigen-binding fragment thereof comprising, for example, V H CDR1, CDR2, and CDR3 sequences and V L CDR1, CDR2, and CDR3 sequences of any one of the presently disclosed scFv (e.g., 3-P14, 4-B2, 1-J19-2, 1-P13, 1-P23, 1-a20, 2-N3, 1-H19, 2-F18, and 4-P3) to bind to CD33 (e.g., human CD 33). In certain embodiments, the extracellular antigen-binding domain of the presently disclosed CAR cross-competes with a reference antibody or antigen-binding portion thereof comprising, for example, the V H sequence and V L sequence of any of the presently disclosed scFv (e.g., 3-P14, 4-B2, 1-J19-2, 1-P13, 1-P23, 1-a20, 2-N3, 1-H19, 2-F18, and 4-P3) for binding to CD33 (e.g., human CD 33).
In certain embodiments, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) cross-competes for binding to CD33 (e.g., human CD 33) with a reference antibody or antigen-binding portion thereof comprising V H CDR1, CDR2, and CDR3 sequences of scFv 3-P14 and V L CDR1, CDR2 and CDR3 sequences. For example, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) cross-competes with a reference antibody or antigen-binding portion thereof that comprises: v H CDR1 comprising the amino acid sequence shown in SEQ ID No. 2; v H CDR2 comprising the amino acid sequence shown in SEQ ID No. 3; V H CDR3 comprising the amino acid sequence set forth in SEQ ID No. 4; v L CDR1 comprising the amino acid sequence shown in SEQ ID No. 5; v L CDR2 comprising an amino acid having the amino acid sequence shown in SEQ ID No. 6; And V L CDR3 comprising an amino acid having the amino acid sequence shown in SEQ ID NO. 7. In certain embodiments, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) cross-competes with a reference antibody or antigen-binding portion thereof comprising V H and V L sequences of scFv 3-P14 for binding to CD33 (e.g., human CD 33). for example, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) cross-competes with a reference antibody or antigen-binding portion thereof that comprises: v H comprising an amino acid having the amino acid sequence set forth in SEQ ID No. 8; and V L comprising an amino acid having the amino acid sequence shown in SEQ ID NO. 9.
In certain embodiments, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) binds to the same epitope region on CD33 (e.g., human CD 33) with a reference antibody or antigen-binding portion thereof. For example, the extracellular antigen-binding domain of the presently disclosed CAR binds to the same epitope region on CD33 (e.g., human CD 33) as a reference antibody or antigen-binding portion thereof comprising, for example, V H CDR1, CDR2, and CDR3 sequences and V L CDR1, CDR2, and CDR3 sequences of any one of the presently disclosed scFv (e.g., 3-P14, 4-B2, 1-J19-2, 1-P13, 1-P23, 1-a20, 2-N3, 1-H19, 2-F18, and 4-P3). In certain embodiments, the extracellular antigen binding domain of the presently disclosed antigen recognizing receptor (e.g., CAR) binds to the same epitope region on CD33 (e.g., human CD 33) as a reference antibody or antigen binding portion thereof comprising, for example, the V H and V L sequences of any of the presently disclosed scFv (e.g., 3-P14, 4-B2, 1-J19-2, 1-P13, 1-P23, 1-a20, 2-N3, 1-H19, 2-F18, and 4-P3).
In certain embodiments, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) cross-competes with a reference antibody or antigen-binding portion thereof for binding to CD33 (e.g., human CD 33). For example, the extracellular antigen binding domain of the presently disclosed antigen recognizing receptor (e.g., CAR) cross-competes with a reference antibody or antigen binding portion thereof comprising, for example, V H CDR1, CDR2, and CDR3 sequences and V L CDR1, CDR2, and CDR3 sequences of any one of the presently disclosed scFv (e.g., 3-P14, 4-B2, 1-J19-2, 1-P13, 1-P23, 1-a20, 2-N3, 1-H19, 2-F18, and 4-P3) to bind to CD33 (e.g., human CD 33). In certain embodiments, the extracellular antigen binding domain of the presently disclosed antigen recognizing receptor (e.g., CAR) binds to the same epitope region on CD33 (e.g., human CD 33) as a reference antibody or antigen binding portion thereof comprising, for example, the V H and V L sequences of any of the presently disclosed scFv (e.g., 3-P14, 4-B2, 1-J19-2, 1-P13, 1-P23, 1-a20, 2-N3, 1-H19, 2-F18, and 4-P3).
Extracellular antigen binding domains that cross-compete or compete for binding to CD33 (e.g., human CD 33) with a reference antibody or antigen binding portion thereof can be identified by using conventional methods known in the art, including, but not limited to, ELISA, radioimmunoassay (RIA), biacore, flow cytometry, western blot, and any other suitable quantitative or qualitative antibody binding assay. Competition ELISA is described in Morris,"Epitope Mapping of ProteinAntigens by Competition ELISA",The Protein Protocols Handbook(1996), pages 595-600, j.walker edit, which is incorporated by reference in its entirety. In certain embodiments, the antibody binding assay comprises measuring initial binding of the reference antibody to the CD33 polypeptide, mixing the reference antibody with the test extracellular antigen binding domain, measuring secondary binding of the reference antibody to the CD33 polypeptide in the presence of the test extracellular antigen binding domain, and comparing the initial binding to the secondary binding of the reference antibody, wherein a decrease in the secondary binding of the reference antibody to the CD33 polypeptide as compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody for binding to CD33, e.g., recognizes the same or substantially the same epitope, overlapping epitope, or adjacent epitope. In certain embodiments, the reference antibody is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD33 polypeptide is expressed in a cell, e.g., in a flow cytometry test. In certain embodiments, the CD33 polypeptide is immobilized to a surface that includes a Biacore chip (e.g., in a Biacore test), or other medium suitable for surface plasmon resonance analysis. In the presence of completely unrelated antibodies (which do not bind to CD 33), the binding of the reference antibody may serve as a control high value. Control low values can be obtained by incubating a labeled reference antibody with an unlabeled reference antibody, wherein binding of the labeled reference antibody competes and decreases. In certain embodiments, reducing binding of the reference antibody to the CD33 polypeptide by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the test extracellular antigen-binding domain is considered to cross-compete with the reference antibody for extracellular antigen-binding domain that binds to CD 33. In certain embodiments, the assay is performed at room temperature.
In certain embodiments, the antibody binding assay comprises measuring initial binding of the test extracellular antigen binding domain to the CD33 polypeptide, mixing the test extracellular antigen binding domain with a reference antibody, measuring secondary binding of the test extracellular antigen binding domain to the CD33 polypeptide in the presence of the reference antibody, and comparing the initial binding to the secondary binding of the test extracellular antigen binding domain, wherein a decrease in the secondary binding of the test extracellular antigen binding domain to the CD33 polypeptide as compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody for binding to CD33, e.g., recognizes the same or substantially the same epitope, overlapping epitope, or adjacent epitope. In certain embodiments, the test extracellular antigen binding domain is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD33 polypeptide is expressed in a cell, e.g., in a flow cytometry test. In certain embodiments, the CD33 polypeptide is immobilized to a surface that includes a Biacore chip (e.g., in a Biacore test), or other medium suitable for surface plasmon resonance analysis. In the presence of completely unrelated antibodies (which do not bind to CD 33), the binding of the test extracellular antigen binding domain may serve as a control high value. By incubating the labeled test extracellular antigen binding domain with the unlabeled test extracellular antigen binding domain, a control low value can be obtained in which the binding of the labeled test extracellular antigen binding domain competes and decreases. In certain embodiments, a test extracellular antigen-binding domain that reduces binding to a CD33 polypeptide by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in the presence of a reference antibody is considered to cross-compete with the reference antibody for extracellular antigen-binding domain that binds to CD 33. In certain embodiments, the assay is performed at room temperature.
In certain embodiments, the extracellular antigen-binding domain of the presently disclosed antigen-recognizing receptor (e.g., CAR) comprises a linker connecting the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 95. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 96. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 97. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 98. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 99. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in: SEQ ID NO. 100.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the heavy chain variable region (V H) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v H-VL.
In certain embodiments, the variable regions within the extracellular antigen-binding domain must be linked one after the other such that the light chain variable region (V L) is located at the N-terminus of the extracellular antigen-binding domain. In certain embodiments, if the extracellular antigen-binding domain is an scFv, the variable region is positioned from N-terminus to C-terminus: v L-VH.
5.3.2. Chimeric Antigen Receptor (CAR)
In certain embodiments, the antigen recognizing receptor is a CAR. CARs are engineered receptors that graft or confer a specific graft of interest to immune effector cells. CARs may be used to graft specificity of monoclonal antibodies onto T cells; transfer of its coding sequence is facilitated by a retroviral vector.
There are three generations of CARs. A "first generation" CAR is typically composed of an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular signaling domain. The "first generation" CARs can provide de novo antigen recognition and activate both CD4 + and CD8 + T cells through the cd3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. A "second generation" CAR adds intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX, 40) to the cytoplasmic tail of the CAR to provide additional signals to T cells. "second generation" CARs include those that provide both co-stimulation (e.g., CD28 or 4-1 BB) and activation (cd3ζ). "third generation" CARs include those that provide both a variety of co-stimuli (e.g., CD28 and 4-1 BB) and activation (cd3ζ). In certain embodiments, the antigen recognizing receptor is a first generation CAR. In certain embodiments, the antigen recognizing receptor is a CAR that does not include an intracellular signaling domain of a costimulatory molecule, or fragment thereof. In certain embodiments, the antigen recognizing receptor is a second generation CAR.
In certain embodiments, the CAR comprises an extracellular antigen binding domain that specifically binds to CD33, a transmembrane domain, and an intracellular signaling domain.
Extracellular antigen binding domain of car
The extracellular antigen-binding domain of the CAR can be any extracellular antigen-binding domain disclosed in section 5.3.1 of this Wen Liru.
In certain embodiments, the CAR comprises an extracellular antigen-binding domain disclosed in section 5.3.1.
In addition, the extracellular antigen-binding domain may include a leader peptide or signal peptide that directs the nascent protein into the endoplasmic reticulum. The CAR is glycosylated and anchored in the cell membrane, a signal peptide or leader peptide may be necessary. The signal sequence or leader sequence may be a peptide sequence (e.g., about 5, about 10, about 15, about 20, about 25, or about 30 amino acids) present at the N-terminus of the newly synthesized protein that directs the protein into the secretory pathway. In certain embodiments, the signal peptide is covalently attached to the 5' end of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises or consists of amino acids 1 to 18 of: SEQ ID NO. 101. An exemplary nucleotide sequence encoding amino acids 1 to 18 of SEQ ID NO. 101 is shown in SEQ ID NO. 122, which is provided below.
ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCA[SEQ ID NO:122]
Transmembrane domain of car 5.3.2.2
In certain embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains produce different receptor stabilities. Following antigen recognition, the receptor aggregates and signals are transmitted to the cell. According to the presently disclosed subject matter, the transmembrane domain of the CAR may include a native or modified transmembrane domain of CD8 or a fragment thereof, a native or modified transmembrane domain of CD28 or a fragment thereof, a native or modified transmembrane domain of CD3 ζ or a fragment thereof, a native or modified transmembrane domain of CD4 or a fragment thereof, a native or modified transmembrane domain of 4-1BB or a fragment thereof, a native or modified transmembrane domain of OX40 or a fragment thereof, a native or modified transmembrane domain of ICOS or a fragment thereof, a native or modified transmembrane domain of CD84 or a fragment thereof, a native or modified transmembrane domain of CD166 or a fragment thereof, a native or modified transmembrane domain of CD8a or a fragment thereof, a native or modified transmembrane domain of CD8b or a fragment thereof, a native or modified transmembrane domain of ICAM-1 or a fragment thereof, a native or modified transmembrane domain of ctlgd-4 or a fragment thereof, a native or a modified transmembrane domain of CD27 or a fragment thereof, a modified transmembrane domain of CD40 or a fragment thereof, a native or a combination thereof.
In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or fragment thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD8 or a fragment thereof. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is as defined in NCBI reference: the amino acid sequence of NP-001139345.1 (SEQ ID NO: 101) or a fragment thereof is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 101, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 137 to 209, or 200 to 235: SEQ ID NO. 101. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137 to 209 of: SEQ ID NO. 101.SEQ ID NO 101 is provided below.
MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLY
LSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV[SEQ ID NO:101]
In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD8 or a fragment thereof. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is as defined in NCBI reference: AAA92533.1 (SEQ ID NO: 102) or a fragment thereof is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical, and/or may optionally contain up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 102, which is at least 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247: SEQ ID NO. 102. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151 to 219 of: SEQ ID NO:102.SEQ ID NO. 102 is provided below.
In certain embodiments, the transmembrane domain of the presently disclosed CARs comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a fragment thereof).
In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a fragment thereof. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is as defined in NCBI reference: the amino acid sequence of NP-006130 (SEQ ID No: 103) or fragments thereof is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 103, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 153 to 179, or 200 to 220 of seq id no: SEQ ID NO. 103. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of: SEQ ID NO. 103.SEQ ID NO. 103 provides the following:
An exemplary nucleotide sequence encoding amino acids 153 to 179 of SEQ ID NO. 103 is shown in SEQ ID NO. 104, which is provided below.
TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG[SEQ ID NO:104]
In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of mouse CD28 or a fragment thereof). In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is as defined in NCBI reference: the amino acid sequence of NP-031668.3 (SEQ ID No: 105) or a fragment thereof is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 105, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 177, or 200 to 218: SEQ ID NO. 105. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151 to 177 of: SEQ ID NO. 105.SEQ ID NO 105 provides as follows:
In certain non-limiting embodiments, the CAR further comprises a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be sufficiently flexible to allow the antigen binding domains to be oriented in different directions, thereby facilitating antigen recognition while maintaining the activation activity of the CAR.
In certain embodiments, the hinge/spacer region of the CAR comprises a native or modified hinge region of CD8 or a fragment thereof, a native or modified hinge region of CD28 or a fragment thereof, a native or modified hinge region of CD3 ζ or a fragment thereof, a native or modified hinge region of CD40 or a fragment thereof, a native or modified hinge region of 4-1BB or a fragment thereof, a native or modified hinge region of OX40 or a fragment thereof, a native or modified hinge region of CD84 or a fragment thereof, a native or modified hinge region of CD166 or a fragment thereof, a native or modified hinge region of CD8a or a fragment thereof, a native or modified hinge region of CD8b or a fragment thereof, a native or modified hinge region of ICOS or a fragment thereof, a native or modified hinge region of ICAM-1 or a fragment thereof, a native or modified hinge region of CTLA-4 or a fragment thereof, a native or modified hinge region of CD27 or a fragment thereof, a native or modified hinge region of CD40 or a fragment thereof, a native or a modified hinge region of nk2 or a fragment thereof, a native or a fragment thereof, a polypeptide that is not associated with a native or a modified protein of gd or a fragment thereof (based on a native or a polypeptide of interest or a combination thereof). The hinge/spacer region may be a hinge region from IgG1 or a CH 2CH3 region of an immunoglobulin as well as portions of CD3, a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO:101 or 102), a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO:103 or 105), a variant of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.
5.3.2.3 Intracellular signaling domain of CAR
In certain embodiments, the CAR comprises an intracellular signaling domain. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a cd3ζ polypeptide. Cd3ζ may activate or stimulate cells (e.g., cells of the stranguria line, such as T cells). Wild-type ("native") CD3 zeta includes three functional immune receptor tyrosine-based activation motifs (ITAMs), three functional base-rich stretch (BRS) regions (BRS 1, BRS2 and BRS 3). After the antigen is bound, cd3ζ transmits an activation signal to cells (e.g., cells of the stranguria line, such as T cells). The intracellular signaling domain of the cd3ζ chain is the primary transmitter of the signal of endogenous TCRs.
In certain embodiments, the intracellular signaling domain of the CAR comprises native cd3ζ. In certain embodiments, the cd3ζ polypeptide comprises or consists of the amino acid sequence of seq id no: the amino acid sequence is as defined in NCBI reference: the amino acid sequence of NP-932170 (SEQ ID NO: 106) or a fragment thereof is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical and/or may optionally contain up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the cd3ζ polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 106, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. In certain embodiments, the cd3ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164: SEQ ID NO. 106. In certain embodiments, the intracellular signaling domain of the CAR comprises or consists of a cd3ζ polypeptide comprising amino acids 52 to 164 of: SEQ ID NO. 106.SEQ ID NO 106 provides as follows:
In certain embodiments, the intracellular signaling domain of the CAR comprises or consists of a CD3 polypeptide comprising an amino acid sequence set forth in seq id no: SEQ ID NO. 107.SEQ ID NO:107 is provided below.
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR[SEQ ID NO:107]
An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 107 is set forth in SEQ ID NO. 108, which is provided below.
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC
AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG
CCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC[SEQ ID NO:108]
In certain embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling region. In certain embodiments, the costimulatory signaling region comprises at least one costimulatory molecule, or a fragment thereof. In certain embodiments, the costimulatory signaling region comprises at least one intracellular domain of a costimulatory molecule, or a fragment thereof.
As used herein, a "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or ligand thereof that can provide a highly efficient response of lymphocytes to an antigen. In certain embodiments, the co-stimulatory molecules may provide optimal lymphocyte activation. Non-limiting examples of co-stimulatory molecules include CD28、4-1BB、OX40、ICOS、DAP-10、CD27、CD40、NKGD2、CD2、FN14、HVEM、LTBR、CD28H、TNFR1、TNFR2、BAFF-R、BCMA、TACI、TROY、RANK、CD40、CD27、CD30、EDAR、XEDAR、GITR、DR6 and NGFR, and combinations thereof. The co-stimulatory molecule may bind to a co-stimulatory ligand, which is a protein expressed on the cell surface that produces a co-stimulatory response upon binding to its receptor, i.e., produces an intracellular response that affects stimulation when an antigen recognizing receptor (e.g., chimeric Antigen Receptor (CAR)) binds to its target antigen. As one example, a 4-1BB ligand (i.e., 4-1 BBL) can bind to 4-1BB to provide an intracellular signal that, in combination with the CAR signal, induces effector cell function of CAR + T cells.
In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of a CD28 polypeptide, such as CD28 or a fragment thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, such as the intracellular domain of human CD28 or a fragment thereof. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO. 103, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 103, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 220 of seq id no: SEQ ID NO. 103. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, the CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 180 to 220 of: SEQ ID NO. 103.
An exemplary nucleotide sequence encoding amino acids 180 to 220 of SEQ ID NO. 103 is shown in SEQ ID NO. 109, which is provided below.
AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC[SEQ ID NO:109]
In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide, e.g., the intracellular domain of mouse CD28 or a fragment thereof. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO 104, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence: the amino acid sequence is a contiguous portion of SEQ ID NO. 104, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 150 to 218, 178 to 218, or 200 to 218: SEQ ID NO. 105. In certain embodiments, the co-stimulatory signaling region of the presently disclosed CAR comprises a CD28 polypeptide comprising or consisting of amino acids 178 to 218 of: SEQ ID NO. 105.
In certain embodiments, the costimulatory signaling region of the presently disclosed CARs comprises a CD28 polypeptide comprising a mutated YMNM motif. CD28 is a transmembrane protein that plays a key role in T cell activation by acting as a co-stimulatory molecule. CD28 possesses an intracellular domain that contains intracellular motifs critical for efficient signaling of CD 28. In certain embodiments, the CD28 intracellular domain comprises an intracellular subdomain (also referred to as an "intracellular motif") that modulates the post-TCR stimulation signaling pathway. CD28 includes three intracellular motifs: YMNM motif and two proline-rich motifs: PRRP motif and PYAP motif. The CD28 intracellular motifs can serve as docking sites for a number of adapter molecules that interact with these motifs through their SH2 or SH3 domains. Such interactions transduce downstream signals, terminating in transcription factors that regulate gene expression. For example, the native YMNM motif binds to the p85 subunit of phosphoinositide 3-kinase (PI 3K). The native YMNM motif also binds to growth factor receptor binding protein 2 (Grb 2) and/or Grb 2-associated adaptor protein 2 (GADS). Grb2 binds to Gab1 and Gab2, and can recruit the p85 subunit of PI 3K.
In certain embodiments, the native YMNM motif consists of the amino acid sequence shown in YMNM (SEQ ID NO: 123). In certain embodiments, the native YMNM motif binds to the p85 subunit of PI3K via the consensus sequence YMxM (SEQ ID NO: 124), where x is not aspartic acid (N). In certain embodiments, the native YMNM motif binds to Grb2 and/or GADs via the consensus sequence YxNx (SEQ ID NO: 125), wherein x is not methionine (M).
In certain embodiments, the recruitment of the p85 subunit of PI3K of CD28 polypeptides comprising the mutated YMNM motif of the present disclosure is reduced compared to a CD28 molecule comprising the native YMNM motif. In certain embodiments, the p85 subunit of PI3K does not bind to the mutated YMNM motif, thereby reducing recruitment of CD28 polypeptide by the p85 subunit of PI 3K. The mutated YMNM motif blocking the binding of the p85 subunit of PI3K retains its binding to Grb2 and/or GADS. Thus, the downstream signaling of Grb2/GADS remains intact, e.g., downstream signaling leading to IL-2 secretion remains intact. The YMNM motif of such mutations is referred to as "GADS/Grb2 permissive mutant".
In certain embodiments, the mutated YMNM binds to the p85 subunit of PI3K, but not to Grb2 and/or GADS. Since the binding of PI3K p was preserved, the downstream signaling of PI3K remained intact. Since binding of Grb2/GADS is blocked, recruitment of the PI3K p85 subunit triggered by Grb2 binding to Gab1 and Gab2 is reduced or blocked. In addition, the downstream signaling of Grb2/GADS is blocked. Such mutated YMNM motif is referred to as "PI 3K-permissive mutant".
In certain embodiments, the mutated YMNM does not bind to the p85 subunit of PI3K and does not bind to Grb2 and/or GADS. Such mutated YMNM motif is referred to as "nonfunctional mutant". Non-functional mutants do not provide for binding of PI3K, grb2 or GADS to CD28 at the YMNM motif, but do not exclude binding of these signaling molecules to other positions in the CD28 molecule.
In certain embodiments, the mutated YMNM retains only one of the two methionine residues present in the YMNM motif, i.e., YMxx or YxxM. These motifs potentially regulate signaling through PI3 ks by limiting the number of methionine residues that can bind to the p85 subunit of PI 3K. Such mutated YMNM motif is referred to as a "hybrid 'HEMI' mutant".
In certain embodiments, the YMNM motif of the mutation is GADS/Grb-2 permissive mutant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YxNx (SEQ ID NO: 125), where x is not methionine (M). In certain embodiments, x is selected from the group consisting of: amino acids A, R, N, D, C, E, Q, G, H, I, K, F, P, S, T, W, Y, V and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YENV(SEQ ID NO:126)、YSNV(SEQ ID NO:127)、YKNL(SEQ ID NO:128)、YENQ(SEQ ID NO:129)、YKNI(SEQ ID NO:130)、YINQ(SEQ ID NO:131)、YHNK(SEQ ID NO:132)、YVNQ(SEQ ID NO:133)、YLNP(SEQ ID NO:134)、YLNT(SEQ IDNO:135)、YDND(SEQ ID NO:136)、YENI(SEQ ID NO:137)、YENL(SEQ ID NO:138)、YKNQ(SEQ ID NO:139)、YKNV(SEQ ID NO:140) or YANG (SEQ ID NO: 141). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YSNV (SEQ ID NO: 127). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YKNI (SEQ ID NO: 130). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YENV (SEQ ID NO: 126). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YKNL (SEQ ID NO: 128).
In certain embodiments, the mutated YMNM motif is a PI 3K-permissive mutant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YMxM (SEQ ID NO: 124), where x is not aspartic acid (N). In certain embodiments, x is selected from the group consisting of: amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YMDM (SEQ ID NO: 142), YMPM (SEQ ID NO: 143), YMRM (SEQ ID NO: 144) or YMSM (SEQ ID NO: 145). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YMDM (SEQ ID NO: 142).
In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YbxM (SEQ ID NO: 146), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of: amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V and L. In certain embodiments, b is selected from the group consisting of: amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YTHM (SEQ ID NO: 147), YVLM (SEQ ID NO: 148), YIAM (SEQ ID NO: 149), YVEM (SEQ ID NO: 150), YVKM (SEQ ID NO: 151) or YVPM (SEQ ID NO: 152).
In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YMxb (SEQ ID NO: 153), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of: amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V and L. In certain embodiments, b is selected from the group consisting of: amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YMAP (SEQ ID NO: 154).
Some mutated YMNM motifs are described in Mol Cell proteomics.2010, 11; 9 (11) 2391-404; virology.2015, month 5; 0:568-577, both of which are incorporated herein by reference in their entirety.
In certain embodiments, the mutated YMNM motif is a hybrid 'HEMI' mutant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YMNx (SEQ ID NO: 155) or YxNM (SEQ ID NO: 156), where x is not methionine (M). In certain embodiments, x is selected from the group consisting of: amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMNV (SEQ ID NO: 157), YENM (SEQ ID NO: 158), YMNQ (SEQ ID NO: 159), YMNL (SEQ ID NO: 160) or YSNM (SEQ ID NO: 161).
In certain embodiments, the mutated YMNM motif is a nonfunctional mutant. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in amino acid sequence Ybxb (SEQ ID NO: 162), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of: A. r, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V and L. In certain embodiments, b is selected from the group consisting of: A. r, N, D, C, E, Q, G, H, I, K, F, P, S, T, W, Y, V and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YGGG(SEQ ID NO:163)、YAAA(SEQ ID NO:164)、YFFF(SEQ ID NO:165)、YETV(SEQ ID NO:166)、YQQQ(SEQ ID NO:167)、YHAE(SEQ ID NO:168)、YLDL(SEQ ID NO:169)、YLIP(SEQ IDNO:170)、YLRV(SEQ ID NO:171)、YTAV(SEQ ID NO:172) or YVHV (SEQ ID NO: 173). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence shown in YGGG (SEQ ID NO: 163).
In certain embodiments, the intracellular signaling domain of the presently disclosed chimeric receptor comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif, the mutated YMNM motif consisting of the amino acid sequence shown in YENV (SEQ ID NO: 126), wherein the CD28 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:174.SEQ ID NO:174 is provided below.
RSKRSRLLHSDYENVTPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO:174]
In certain embodiments, the intracellular signaling domain of the presently disclosed chimeric receptor comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif, the mutated YMNM motif consisting of the amino acid sequence shown in YKNI (SEQ ID NO: 130), wherein the CD28 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:175.SEQ ID NO 175 is provided below.
RSKRSRLLHSDYKNITPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO:175]
In certain embodiments, the intracellular signaling domain of the presently disclosed chimeric receptor comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif, the mutated YMNM motif consisting of the amino acid sequence shown in YMDM (SEQ ID NO: 142), wherein the CD28 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:176.SEQ ID NO. 176 is provided below.
RSKRSRLLHSDYMDMTPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO:176]
In certain embodiments, the intracellular signaling domain of the presently disclosed chimeric receptor comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif, the mutated YMNM motif consisting of the amino acid sequence shown in YGGG (SEQ ID NO: 163), wherein the CD28 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:177.SEQ ID NO:177 is provided below.
RSKRSRLLHSDYGGGTPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO:177]
In certain embodiments, the intracellular signaling domain of the presently disclosed chimeric receptor comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif, the mutated YMNM motif consisting of the amino acid sequence shown in YSNV (SEQ ID NO: 127), wherein the CD28 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:178.SEQ ID NO 178 is provided below.
RSKRSRLLHSDYSNVTPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO:178]
In certain embodiments, the intracellular signaling domain of the presently disclosed CARs comprises a first costimulatory signaling domain comprising the CD28 polypeptide comprising the mutated YMNM motif (as disclosed herein) and a second costimulatory signaling domain comprising the intracellular domain of the costimulatory molecule. Additional information regarding CARs comprising a CD28 polypeptide comprising a mutated YMNM motif can be found in international patent publication No. WO 2021/158850, which is incorporated by reference in its entirety.
In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of a 4-1BB polypeptide, e.g., 4-1BB or fragment thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the 4-1BB polypeptide, e.g., the intracellular domain of human 4-1BB or a fragment thereof. In certain embodiments, the 4-1BB polypeptide comprises or consists of the amino acid sequence of seq id no: the amino acid sequence is as defined in NCBI reference: NP-001552 (SEQ ID NO: 110) or a fragment thereof is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the 4-1BB polypeptide comprises or consists of the amino acid sequence of seq id no: the amino acid sequence is a contiguous portion of SEQ ID NO. 106, which is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 150, and up to 255 amino acids in length. In certain embodiments, the 4-1BB polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 255: SEQ ID NO. 110. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide comprising or consisting of the amino acid sequence of amino acids 214 to 255 of: SEQ ID NO. 110.SEQ ID NO 110 is provided below.
An exemplary nucleotide sequence encoding amino acids 214 to 255 of SEQ ID NO. 110 is shown in SEQ ID NO. 111, which is provided below.
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG[SEQ ID NO:111]
In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domains of two or more costimulatory molecules, or portions thereof, such as the intracellular domain of CD28 or fragment thereof and the intracellular domain of 4-1BB or fragment thereof, or the intracellular domain of CD28 or fragment thereof and the intracellular domain of OX40 or fragment thereof.
In certain embodiments, the presently disclosed CARs further comprise an inducible promoter for expressing the nucleic acid sequence in human cells. The promoter used to express the CAR gene can be a constitutive promoter, such as the ubiquitin C (UbiC) promoter.
5.3.2.4. Exemplary CAR
In certain embodiments, the CAR is a CD 33-targeting CAR. In certain embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) V H, comprising: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4, and (ii) V L, it comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO.5, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 6, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 7; (b) A transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a fragment thereof), and (c) an intracellular signaling domain comprising (i) a CD3 ζ polypeptide, and (ii) a costimulatory signaling region comprising the CD28 polypeptide (e.g., an intracellular domain of human CD28 or a fragment thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising amino acids 153 to 179 of: SEQ ID NO. 103. In certain embodiments, the intracellular signaling domain comprises (i) a CD3 ζ polypeptide comprising the amino acid sequence shown in SEQ ID No. 107, and (ii) a costimulatory signaling region comprising a CD28 polypeptide comprising the amino acid sequences amino acids 180-220 of: SEQ ID NO. 103. In certain embodiments, the extracellular antigen-binding domain is an scFv, designated as "3-P14". In certain embodiments, V H and V L are linked via a linker comprising or consisting of the amino acid sequences set forth in seq id no: SEQ ID NO. 96. In certain embodiments, V H and V L are positioned from N-terminus to C-terminus: v H-VL. In certain embodiments, CAR is designated as "TDI-Y-006_h28z". In certain embodiments, the CAR comprises an amino acid sequence set forth in: SEQ ID NO. 112, which provides as follows.
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSAISGRGGSTYYTDSVKGRFTISR
DNSKNTVSLQMNSLRAEDTAVYYCAGRGDYYYYYGMDVWGQGTTVTVSAGGGGSGGGGSGGGGSDIVMTQSP
LSSPVTLGQPASFSCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKI
SRVEAEDVGVYYCMQSTQFPHTFGQGTKLEIKEQKLISEEDLAAAIEVMYPPPYLDNEKSNGTIIHVKGKHL
CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP
RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR[SEQ ID NO:112]
An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 112 is shown in SEQ ID NO. 113, which is provided below.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC
TCTGGATTCACCTTTAGCACCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCAGCTATTAGTGGTCGTGGTGGTAGCACATACTACACAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA
GACAATTCCAAGAACACGGTGTCTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT
GCGGGCCGGGGAGATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC
GCAGGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCTGATATTGTGATGACCCAGAGTCCA
CTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCTTCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGT
GATGGAAACACCTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAAGACTCCTAATTTATAAGATT
TCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATC
AGCAGGGTGGAAGCTGAGGATGTCGGGGTTTATTACTGCATGCAATCTACACAATTTCCTCACACTTTTGGC
CAGGGGACCAAGCTGGAGATCAAAGAACAGAAACTGATCTCTGAAGAAGACCTGGCGGCCGCAATTGAAGTT
ATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTT
TGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCT
TGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCAC
AGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCA
CGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGC
CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC
CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA
GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC
CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAG[SEQ ID NO:113]
In certain embodiments, the CAR is a CD 33-targeting CAR. in certain embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) V H, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and V H CDR3, Comprising the amino acid sequence shown in SEQ ID NO. 14, and (ii) V L comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:15, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and V L CDR3, Which comprises the amino acid sequence shown in SEQ ID NO. 17; (b) A transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof), and (c) an intracellular signaling domain comprising (i) a CD3 ζ polypeptide, and (ii) a costimulatory signaling region comprising the CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising amino acids 153 to 179 of: SEQ ID NO. 103. In certain embodiments, the intracellular signaling domain comprises (i) a CD3 ζ polypeptide comprising the amino acid sequence shown in SEQ ID No. 107, and (ii) a costimulatory signaling region comprising a CD28 polypeptide comprising the amino acid sequences amino acids 180-220 of: SEQ ID NO. 103. In certain embodiments, the extracellular antigen-binding domain is an scFv, which is designated "4-B2". In certain embodiments, V H and V L are linked via a linker comprising or consisting of the amino acid sequences set forth in seq id no: SEQ ID NO. 96. In certain embodiments, V H and V L are positioned from N-terminus to C-terminus: v H-VL. in some embodiments, CAR is designated as "TDI-Y-007_h28z". In certain embodiments, the CAR comprises an amino acid sequence set forth in: SEQ ID NO. 114, which provides as follows.
EVHLLESGGGLVQPGGSLRLSCAASGFIFSSNAMSWVRQAPGKGLEWVSAISGYGGNTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKWGTYIVGATGDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPP
SASGSPGQSVTISCTGTSNDVGGYNYVSWYQQHPGKAPKLLIYEVSKRPSGVPDRFSGSQSGNTASLTVSGL
QAEDEADYYCSSYAGSNNWVFGGGTKLTVLEQKLISEEDLAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCP
SPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD
FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR[SEQ ID NO:114]
An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 114 is set forth in SEQ ID NO. 115, which is provided below.
GAGGTGCACCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC
TCTGGATTCATCTTTAGCAGCAATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTC
TCAGCTATTAGTGGTTATGGTGGTAACACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGA
GACAATTCCAAGAACACGCTATATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT
GCGAAATGGGGGACTTATATAGTGGGAGCTACGGGTGACTACTGGGGCCAGGGAACTCTGGTCACCGTCTCC
TCAGGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCTCAGTCTGCCCTGACTCAGCCTCCC
TCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAATGACGTTGGTGGTTAT
AACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCTTGATTTATGAGGTCAGTAAGCGG
CCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCCAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTC
CAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAACAATTGGGTGTTCGGCGGAGGG
ACCAAGCTGACCGTCCTAGAACAGAAACTGATCTCTGAAGAAGACCTGGCGGCCGCAATTGAAGTTATGTAT
CCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCA
AGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTAT
AGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGAC
TACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGAC
TTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC
CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC
CCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAG
ATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC
CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAG
[SEQ ID NO:115]
In certain embodiments, the CAR is a CD 33-targeting CAR. In certain embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising (i) V H, comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:22, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:23, and V H CDR3, Comprising the amino acid sequence set forth in SEQ ID NO. 24, and (ii) V L comprising: CDR1, the CDR1 comprising the amino acid sequence shown in SEQ ID NO:25, CDR2, the CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and V L CDR3, comprising the amino acid sequence shown in SEQ ID NO. 27; (b) A transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof), and (c) an intracellular signaling domain comprising (i) a CD3 ζ polypeptide, and (ii) a costimulatory signaling region comprising the CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising amino acids 153 to 179 of: SEQ ID NO. 103. In certain embodiments, the intracellular signaling domain comprises (i) a CD3 ζ polypeptide comprising the amino acid sequence shown in SEQ ID No. 107, and (ii) a costimulatory signaling region comprising a CD28 polypeptide comprising the amino acid sequences amino acids 180-220 of: SEQ ID NO. 103. In certain embodiments, the extracellular antigen-binding domain is an scFv, which is designated "1-J19". In certain embodiments, V H and V L are linked via a linker comprising or consisting of the amino acid sequences set forth in seq id no: SEQ ID NO. 96. In certain embodiments, V H and V L are positioned from N-terminus to C-terminus: v H-VL. In certain embodiments, CAR is designated as "1j19hl_hz". In certain embodiments, the CAR comprises an amino acid sequence set forth in: SEQ ID NO. 116, which provides as follows.
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYFRSKWYNVYAVSVKSRIT
INPDTSKNQFSLQLNSVTPEDTAVYYCASEGGSYYDHWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPS
SVSASVGDRVTITCRASQGISNWLTWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQADSFPFTFGPGTKVDIKEQKLISEEDLAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLF
PGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY
RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR[SEQ ID NO:116]
An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 116 is set forth in SEQ ID NO. 117, which is provided below.
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATC
TCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAG
TGGCTGGGAAGGACATACTTCAGGTCCAAGTGGTATAATGTTTATGCAGTGTCTGTGAAGAGTCGAATAACC
ATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTG
TATTATTGTGCAAGCGAGGGTGGGAGCTATTATGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
GGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATCTGACATCCAGATGACCCAGTCTCCATCT
TCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGTAATTGGTTA
ACCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG
GTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAA
GATTTTGCAACTTACTATTGTCAACAGGCTGACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGAT
ATCAAAGAACAGAAACTGATCTCTGAAGAAGACCTGGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTAC
CTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTT
CCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTA
ACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATG
ACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTAT
CGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAAC
GAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG
GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC
TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAG[SEQ ID NO:117]
In certain embodiments, the presently disclosed CARs further comprise an inducible promoter for expressing the nucleotide sequence in human cells. The promoter used to express the CAR gene can be a constitutive promoter, such as the ubiquitin C (UbiC) promoter.
Tcr-like fusion molecules
In certain embodiments, the antigen recognizing receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also referred to as "HIT-CARs", such as those disclosed in international patent application No. PCT/US19/017525, which is incorporated by reference in its entirety), T cell receptor fusion constructs (TRuC) (e.g., volume 10 of Baeuerle et al ,"Synthetic TRuC receptorsengaging the complete T cell receptor for potent anti-tumor response,"NatureCommunications, article No. 2087 (2019), which is incorporated by reference in its entirety), and T cell antigen coupling agents (TACs), which are chimeric receptors that sequester endogenous TCRs (e.g., those disclosed in Helsen et al ,"The chimeric TAC receptor co-opts the T cell receptor yielding robustanti-tumor activity without toxicity,"Nature Communications(2018);9:3049(2018)), which is incorporated by reference in its entirety.
In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an extracellular antigen-binding domain and a constant domain, wherein the TCR-like fusion molecule binds to an antigen in an HLA-independent manner. In certain embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of: natural or modified TRAC peptide, natural or modified TRBC peptide, natural or modified TRDC peptide, natural or modified TRGC peptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC peptide. in certain embodiments, the constant domain comprises a native or modified TRBC peptide. In certain embodiments, a constant domain is capable of forming a homodimer or a heterodimer with another constant domain. In certain embodiments, the antigen binding chain is capable of associating with a cd3ζ polypeptide. In certain embodiments, the antigen binding chain is capable of activating a cd3ζ polypeptide associated with the antigen binding chain upon binding to an antigen. In certain embodiments, activation of the cd3ζ polypeptide is capable of activating an immune response cell. In certain embodiments, the TCR-like fusion molecule is capable of integrating with the CD3 complex and providing HLA-independent antigen recognition. in certain embodiments, the TCR-like fusion molecule replaces an endogenous TCR in the CD3/TCR complex. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion of an antibody or fragment thereof, or an antigen-binding portion of a TCR. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises one or two immunoglobulin variable regions. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the heavy chain variable region (V H) of the antibody. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the light chain variable region (V L) of the antibody. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises V H of the antibody, wherein V H is capable of dimerizing with another extracellular antigen-binding domain comprising antibody V L and forming a variable fragment (Fv). In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises V L of the antibody, wherein V L is capable of dimerizing with another extracellular antigen-binding domain comprising antibody V H and forming a variable fragment (Fv).
5.4. Cells
The presently disclosed subject matter provides cells comprising the presently disclosed CD 33-targeting antigen recognizing receptor (e.g., the antigen recognizing receptor disclosed in section 5.3). In certain embodiments, the cell is selected from the group consisting of: the stem cells from which the gonococcal cells, the myeloid cells and the stem cells from which the myeloid cells can be derived. In certain embodiments, the cell is an immune response cell. In certain embodiments, the immune response cell is a gonococcal cell.
In certain embodiments, the cells are cells of a stranguria line. Cells of the stranguria line can produce antibodies, modulate the cellular immune system, detect foreign agents in the blood, detect foreign cells of the host, and the like. Non-limiting examples of cells of the lineage include T cells, natural Killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells may be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell).
In certain embodiments, the cell is a T cell. T cells may be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. T cells of the presently disclosed subject matter may be any type of T cells, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells), stem cell-like memory T cells (or stem-like memory T cells), and effector memory T cells of the following two types: for example, TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), tumor Infiltrating Lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing death of infected somatic or tumor cells. By introducing antigen recognizing receptors, such as CARs, T cells of the patient themselves can be genetically modified to target specific antigens. In certain embodiments, the immune response cell is a T cell. The T cells may be CD4 + T cells or CD8 + T cells. In certain embodiments, the T cell is a CD4 + T cell. In certain embodiments, the T cell is a CD8 + T cell.
In certain embodiments, the cell is an NK cell. Natural Killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and function during an innate immune response. NK cells do not require prior activation to produce cytotoxic effects on target cells.
Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes. For example Sadelain et al, NAT REV CANCER (2003); 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), morgan, r.a., et al 2006Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full length tumor antigen recognition T cell receptor complexes comprising alpha and beta heterodimers), panelli et al, J Immunol (2000); 164:495-504; panelli et al, J Immunol (2000); 164:4382-4392 (discloses lymphocyte cultures derived from Tumor Infiltrating Lymphocytes (TILs) in tumor biopsies), and Dupont et al CANCER RES (2005); 65:5417-5427; papanicolaou et al, blood (2003); 102:2498-2505 (discloses antigen-specific peripheral blood leukocytes selectively expanded in vitro using Artificial Antigen Presenting Cells (AAPC) or pulsed dendritic cells).
Cells (e.g., T cells) may be autologous, non-autologous (e.g., allogeneic) or derived in vitro from engineered progenitor or stem cells.
The cells of the presently disclosed subject matter may be myeloid cells. Non-limiting examples of myeloid cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets and stem cells from which the myeloid cells can differentiate. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).
In certain embodiments, the presently disclosed cells are capable of modulating a tumor microenvironment. Tumors have a microenvironment that is detrimental to the host immune response, which involves a series of mechanisms by which malignant cells protect themselves from immune recognition and elimination. Such "adverse tumor microenvironments" include a variety of immunosuppressive factors, including infiltrative regulatory CD4 + T cells (tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines (including TGF- β), and ligand expression targeting immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). These immunosuppressive mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses, however in tumor microenvironments, these mechanisms prevent effective anti-tumor immune responses. Overall, these immunosuppressive factors can induce significant anergy or apoptosis of adaptively transferred CAR-modified T cells upon encountering targeted tumor cells.
In certain embodiments, the cells may be transduced with the presently disclosed CD 33-targeting antigen recognizing receptor such that the cells express the antigen recognizing receptor.
In certain embodiments, the cell further comprises a soluble single chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive or immunostimulatory activity. In certain embodiments, immunosuppressive activity refers to induction of signal transduction or changes in protein expression in a cell (e.g., an activated immune response cell), thereby causing a decrease in immune response. Polypeptides known to inhibit or reduce an immune response via their binding include CD47, PD-1, CTLA-4 and their corresponding ligands, including SIRPa, PD-L1, PD-L2, B7-1 and B7-2. Such polypeptides are present in the tumor microenvironment and inhibit an immune response to tumor cells. In various embodiments, inhibiting, blocking or antagonizing the interaction of an immunosuppressive polypeptide and/or its ligand enhances the immune response of an immunoresponsive cell.
In certain embodiments, immunostimulatory activity refers to induction of signal transduction or a change in protein expression in a cell (e.g., an activated immune response cell), thereby causing an increase in an immune response. Immunostimulatory activity may include pro-inflammatory activity. Polypeptides known to stimulate or enhance an immune response via their binding include CD28, OX-40, 4-IBB and their corresponding ligands, including B7-1, B7-2, OX-40L and 4-1BBL. Such polypeptides are present in the tumor microenvironment and activate an immune response to tumor cells. In various embodiments, promoting, stimulating or agonizing the pro-inflammatory polypeptide and/or ligand thereof enhances the immune response of the immunoresponsive cell.
Cells comprising an antigen recognizing receptor (e.g., CAR) and a soluble scFv that binds to a polypeptide having immunosuppressive or immunostimulatory activity are disclosed in international patent publication No. WO 2014/134165, which is incorporated by reference in its entirety.
In certain embodiments, the cell further comprises exogenous CD40L. Cells comprising an antigen recognizing receptor (e.g., CAR) and exogenous CD40L are disclosed in international patent publication No. WO 2014/134165.
Furthermore, in certain embodiments, the cells are engineered to express IL-18. In certain embodiments, the cell further comprises an exogenous IL-18 polypeptide. In certain embodiments, the exogenous IL-18 polypeptide comprises or consists of an amino acid sequence set forth in seq id no: SEQ ID NO. 118, which provides as follows.
MGYRMQLLSCIALSLALVTNSGYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIIS
MYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED[SEQ ID NO:118]
In certain embodiments, the cell further comprises a nucleic acid molecule encoding an IL-18 polypeptide. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in: SEQ ID NO:119, which is provided below.
ATGGGTTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGGCTACTTT
GGCAAGCTTGAATCTAAATTATCAGTCATAAGAAATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAAT
CGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTATTATAAGT
ATGTATAAAGATAGCCAGCCTAGAGGTATGGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAACTCTC
TCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATCAAGGATACAAAAAGTGAC
ATCATATTCTTTCAGAGAAGTGTCCCAGGACATGATAATAAGATGCAATTTGAATCTTCATCATACGAAGGA
TACTTTCTAGCTTGTGAAAAAGAGAGAGACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTCACTGTTCAAAACGAAGACTAG[SEQ ID NO:119]
Alternatively, in certain embodiments, the cells further comprise a modified promoter/enhancer at the IL-18 locus that can increase IL-18 gene expression, e.g., a constitutive or inducible promoter is placed to drive IL-18 gene expression.
Cells comprising an antigen recognizing receptor (e.g., CAR) and engineered to express IL-18 (e.g., comprising an exogenous IL-18 polypeptide or a modified promoter/enhancer at the IL-18 locus) are disclosed in international patent publication No. WO2018/027155, which is incorporated by reference in its entirety.
Additionally or alternatively, the cells are engineered to express IL-33. In certain embodiments, the cell further comprises an exogenous IL-33 polypeptide. In certain embodiments, the exogenous IL-33 polypeptide comprises or consists of an amino acid sequence set forth in seq id no: SEQ ID NO. 120, which provides as follows.
MYRMQLLSCIALSLALVTNSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSY
YESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET[SEQ ID NO:120]
In certain embodiments, the cell further comprises a nucleic acid molecule encoding an IL-33 polypeptide. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in: SEQ ID NO. 121, which provides as follows.
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTAGTATCACAGGA
ATTTCACCTATTACAGAGTATCTTGCTTCTCTAAGCACATACAATGATCAATCCATTACTTTTGCTTTGGAG
GATGAAAGTTATGAGATATATGTTGAAGACTTGAAAAAAGATGAAAAGAAAGATAAGGTGTTACTGAGTTAC
TATGAGTCTCAACACCCCTCAAATGAATCAGGTGACGGTGTTGATGGTAAGATGTTAATGGTAACCCTGAGT
CCTACAAAAGACTTCTGGTTGCATGCCAACAACAAGGAACACTCTGTGGAGCTCCATAAGTGTGAAAAACCA
CTGCCAGACCAGGCCTTCTTTGTCCTTCATAATATGCACTCCAACTGTGTTTCATTTGAATGCAAGACTGAT
CCTGGAGTGTTTATAGGTGTAAAGGATAATCATCTTGCTCTGATTAAAGTAGACTCTTCTGAGAATTTGTGTACTGAAAATATCTTGTTTAAGCTCTCTGAAACTTAG[SEQ ID NO:121]
Alternatively, in certain embodiments, the cells further comprise a modified promoter/enhancer at the IL-33 locus that can increase IL-33 gene expression, e.g., a constitutive or inducible promoter placed to drive IL-33 gene expression. Cells comprising an antigen recognizing receptor (e.g., CAR) and engineered to express IL-33 (e.g., comprising an exogenous IL-33 polypeptide or a modified promoter/enhancer at the IL-33 locus) are disclosed in international patent publication No. WO2019/099479, which is incorporated by reference in its entirety.
Additionally or alternatively, the cells are engineered to express IL-36. In certain embodiments, the cell further comprises an exogenous IL-36 polypeptide. In certain embodiments, the cells further comprise a modified promoter/enhancer at the IL-36 locus that can increase IL-36 gene expression, e.g., a constitutive or inducible promoter positioned to drive IL-36 gene expression. Cells comprising an antigen recognizing receptor (e.g., CAR) and engineered to express IL-36 (e.g., comprising an exogenous IL-36 polypeptide or a modified promoter/enhancer at the IL-36 locus) are disclosed in international patent publication No. WO2019/099483, which is incorporated by reference in its entirety.
5.5. Nucleic acid compositions and vectors
The presently disclosed subject matter provides a nucleic acid encoding a presently disclosed CD 33-targeting antigen recognizing receptor (e.g., the antigen recognizing receptor disclosed in section 5.3). Further provided are nucleic acid compositions comprising the nucleic acids disclosed herein. Also provided are cells comprising such nucleic acid compositions.
In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the presently disclosed CD 33-targeting antigen recognizing receptor.
In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the group consisting of an Elongation Factor (EF) -1 promoter, a cytomegalovirus immediate early promoter (CMV) promoter, a simian virus 40 early promoter (SV 40) promoter, a phosphoglycerate kinase (PGK) promoter, and a metallothionein promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of the NFAT Transcription Response Element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter. The compositions and nucleic acid compositions can be administered to a subject or/and delivered into cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be accomplished by transducing a substantially homogenous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector) is used to introduce the DNA construct into a cell. For example, polynucleotides encoding antigen recognizing receptors can be cloned into retroviral vectors and expression can be driven by their endogenous promoters, retroviral long terminal repeats, or promoters specific for the target cell type of interest. Non-viral vectors may also be used.
To initially genetically modify a cell to include the presently disclosed CD 33-targeting antigen recognition receptor (e.g., CAR), retroviral vectors can be employed for transduction, although any other suitable viral vector or non-viral delivery system can be used. The antigen recognizing receptor may be constructed in a single polycistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1IRES, FGF-2IRES, VEGF IRES, IGF-II IRES, NF- κB IRES, RUNX 1IRES, P53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphtha virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A peptides, T2A peptides, E2A peptides, and F2A peptides). Combinations of retroviral vectors and suitable packaging systems are also suitable, wherein the capsid protein will function to infect human cells. A variety of Cell lines are known to produce a compatible virus, including but not limited to PA12 (Miller et al, (1985) Mol Cell Biol (1985); 5:431-437); PA317 (Miller et al Mol Cell Biol (1986); 6:2895-2902); and CRIP (Danos et al, proc NATL ACAD SCI USA (1988); 85:6460-6464). Non-ampholytic particles are also suitable, for example, enveloped with VSVG, RD114 or GALV and any other particle pseudotype known in the art.
Possible transduction methods also include direct co-culture of cells with producer cells (Bregni et al, blood (1992); 80:1418-1422), or with viral supernatant alone or concentrated carrier stock with or without appropriate growth factors and polycations (Xu et al, exp Hemat (1994); 22:223-230; and Hughes et al J CLIN INVEST (1992); 89:1817).
Other transduced viral vectors can be used to modify cells. In certain embodiments, the selected vectors exhibit efficient infection and stable integration and expression (see, e.g., cayouette et al, human GENE THERAPY 8:423-430,1997; kido et al, current EYE RESEARCH 15:833-844,1996; bloom et al, journal ofVirology 71:6641-6649,1997; naldin et al, science 272:263-267,1996; and Miyoshi et al, proc. Natl. Acad. Sci. U.S. A.94:10319,1997). Other viral vectors that may be used include, for example, adenovirus, lentivirus and adenine-related viral vectors, vaccinia virus, bovine papilloma virus or herpes virus, such as Epstein-Barr virus (see also, for example, vectors in Miller, human GENE THERA (1990); 15-14;Friedman,Science 244:1275-1281,1989; eglitis et al, bioTechniques (1988); 6:608-614; tolstoshav et al, cur Opin Biotechnol (1990); 1:55-61;Sharp,The Lancet (1991); 337:1277-78; cornetta et al ,Nucleic Acid Research and Molecular Biology 36:311-22,1987;Anderson,Science(1984);226:401-409;Moen,Blood Cells 17:407-16,1991;Miller et al, biotechnol (1989); 7:980-90;LeGal La Salle et al, science (1993); 259:988-90; hnson, chest (1995) 107:77S-83S). Retroviral vectors are particularly mature and have been used in clinical settings (Rosenberg et al, N Engl J Med (1990); 323:370,1990; anderson et al, U.S. Pat. No. 5,399,346).
Non-viral methods may also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering the nucleic acid in the presence of lipofection (Feigner et al, proc NATL ACAD SCI U.S. A. (1987); 84:7413; ono et al, neurosci Lett (1990); 17:259; brigham et al, am J Med Sci (1989); 298:278; staubinger et al, methods inEnzymol (1983); 101:512, wu et al, J Biol Chem (1988); 263:14621; wu et al, J Biol Chem (1989); 264:16985), or by microinjection under surgical conditions (Wolff et al, science (1990); 247:1465). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes can also have potential benefits for delivery of DNA into cells. Transplanting the normal gene into the affected tissue of the subject may also be accomplished by ex vivo transfer of the normal nucleic acid into a cell type that can be cultured (e.g., autologous or heterologous primary cells or progeny thereof), followed by injection of the cells (or their progeny) into the targeted tissue or systemic injection. Recombinant receptors can also be derived or obtained using transposases or targeting nucleases (e.g., zinc finger nucleases, meganucleases or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.
Any targeted genome editing method can also be used to deliver the presently disclosed antigen recognizing receptors to cells or subjects. In certain embodiments, the CRISPR system is used to deliver the presently disclosed antigen recognizing receptors disclosed herein. In certain embodiments, zinc finger nucleases are used to deliver antigen recognition receptors. In certain embodiments, the TALEN system is used to deliver the presently disclosed antigen recognizing receptors.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems are genomic editing tools found in prokaryotic cells. When used for genome editing, the system comprises Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, containing the RNA used by Cas9 to guide it to the correct segment of host DNA, and a region that binds to the tracrRNA (typically in hairpin loop form) to form an active complex with Cas 9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas 9), and an optional segment of DNA repair template (DNA that guides the cell repair process, allowing insertion of specific DNA sequences). CRISPR/Cas9 generally employs plasmids to transfect target cells. crrnas need to be designed for each application, as this is the sequence that Cas9 uses to identify and bind directly to target DNA in cells. Repair templates carrying CAR expression cassettes also need to be designed for each application, as it must overlap with sequences on either side of the cut and encode the insertion sequence. Multiple crrnas and tracrRNA may be packaged together to form a single guide RNA (sgRNA). This sgRNA can be ligated together with the Cas9 gene and made into a plasmid for transfection into cells.
Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes that are produced by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, which allows the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domains of individual ZFNs typically contain multiple individual zinc finger repeats and each can recognize multiple base pairs. The most common method for generating new zinc finger domains should combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type IIs restriction endonuclease fokl. ZFNs can be used to insert CAR expression cassettes into the genome using endogenous Homologous Recombination (HR) mechanisms and homologous DNA templates carrying the CAR expression cassettes. When the targeting sequence is cleaved by ZFNs, the HR mechanism searches for homology between the compromised chromosome and the homologous DNA template, and then copies the sequence of the template between the two cleaved ends of the chromosome, whereby the homologous DNA template is integrated into the genome.
Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific DNA sequences. The principle of operation of TALEN systems is almost the same as ZFNs. They are produced by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. The transcription activator-like effector (TALE) consists of a 33-34 amino acid repeat motif with two variable positions and has strong recognition for specific nucleotides. By assembling these TALE arrays, the TALE DNA binding domains can be engineered to bind to the desired DNA sequence and thereby direct the nuclease to cleave at specific locations in the genome. cDNA expression for use in the polynucleotide treatment methods may be directed by any suitable promoter (e.g., human Cytomegalovirus (CMV), simian Virus 40 (SV 40), or metallothionein promoter) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer/promoter/intron structure). For example, enhancers known to preferentially direct gene expression in a particular cell type can be used to direct expression of a nucleic acid, if desired. Enhancers used may include, but are not limited to, enhancers characterized as tissue or cell specific enhancers. Alternatively, if genomic clones are used as therapeutic constructs, modulation may be mediated by homologous regulatory sequences or, if desired, by regulatory sequences derived from heterologous sources comprising any of the promoters or regulatory elements described above.
5.5.1 Delivery methods the method used to deliver the genome editing agent/system can vary as desired. In certain embodiments, components of the selected genome editing methods are delivered as DNA constructs in one or more plasmids. In certain embodiments, these components are delivered by a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, puncture, hydrostatic pressure, continuous infusion, sonication, magnetic infection, adeno-associated viruses, envelope protein pseudotyped viral vectors, replicative vector cis-and trans-acting elements, herpes simplex viruses, and chemical mediators (e.g., oligonucleotides, liposome complexes (lipoplex), polymer vesicles, polyplexes, dendrimers, inorganic nanoparticles, and cell penetrating peptides).
In certain embodiments, the delivery method comprises using a colloid. As used herein, the term "colloid" refers to a system in which two or more phases are present, with one phase (e.g., a dispersed phase) being distributed in the other phase (e.g., a continuous phase). Furthermore, at least one of the phases has a small size (in the range of about 10 -9 to about 10 -6 m). Non-limiting examples of colloids encompassed by the presently disclosed subject matter include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., micelles, liposomes, and lipid nanoparticles).
In certain embodiments, the delivery method comprises using liposomes. As used herein, the term "liposome" refers to a monolayer or multilamellar spherical lipid bilayer structure resulting from lipids dissolved in an organic solvent and then dispersed in an aqueous medium. Experimentally and therapeutically for delivering active pharmaceutical ingredients (e.g., nucleic acid compositions disclosed herein) to cells, liposomes fuse with the cell membrane, and thus the contents are transferred into the cytoplasm.
In certain embodiments, the delivery method comprises using lipid nanoparticles. As used herein, the term "lipid nanoparticle" refers to particles having at least one nanoscale (e.g., about 1nm to about 1,000 nm) size and comprising at least one lipid. In certain embodiments, the lipid nanoparticle may include an active pharmaceutical ingredient (e.g., a nucleic acid composition disclosed herein) for delivery to a cell. The morphology of the lipid nanoparticle may be different from that of a liposome. While liposomes are characterized by a lipid bilayer surrounding a hydrophilic core, lipid nanoparticles have an electron dense core in which cationic lipids and/or ionizable lipids are organized into reverse micelles surrounding the active pharmaceutical ingredient (e.g., a nucleic acid composition as disclosed herein). Additional information regarding the morphology and properties of lipid nanoparticles and liposomes can be found in Wilczewska et al, pharmacological reports 64, stage 5 (2012): 1020-1037; eygeris et al, accounts ofChemical Research, 55, stage 1 (2021): 2-12; zhang et al CHEMICAL REVIEWS, 121, stage 20 (2021): 12181-12277; and Fan et al Journal of pharmaceutical and biomedical analysis 192,192 (2021): 113642.
In certain embodiments, the lipid nanoparticle has an average diameter of about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, about 70nm to about 100nm, about 80nm to about 100nm, about 90nm to about 100nm, about 70 to about 90nm, about 80nm to about 90nm, about 70nm to about 80nm, or about 30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm or 150nm.
In certain embodiments, the lipid nanoparticle may include a cationic lipid or an ionizable lipid. The term "cationic lipid" refers to a lipid comprising a head group bearing a permanent positive charge. Non-limiting examples of cationic lipids encompassed by the presently disclosed subject matter include 1, 2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2, 3-dioleoyloxy-N- [2- (sperminimido) ethyl ] -N, N-dimethyl-1-propanammonium trifluoroacetate (DOSPA), and ethyl phosphatidylcholine (ePC).
As used herein, the term "ionizable lipid" refers to a lipid that is protonated at low pH and neutral at physiological pH. The pH sensitivity of ionizable lipids is particularly advantageous for in vivo delivery (e.g., delivery of the nucleic acid compositions disclosed herein) because neutral lipids interact less with the anionic membrane of blood cells and thus improve the biocompatibility of the lipid nanoparticle. Once trapped in the endosome, the ionizable lipids are protonated and promote membrane destabilization, allowing the nanoparticles to escape the endosome. Non-limiting examples of ionizable lipids encompassed by the presently disclosed subject matter include tetrakis (8-methylnonyl) 3,3',3", 3'" - ((methylazanediyl) bis (propane-3, 1 diyl)) bis (azanetrianyl)) tetrapropionate; decyl (2- (dioctyl ammonium) ethyl) phosphate; ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate); bis (2- (dodecyl-disulfanyl) ethyl) 3,3' - ((3-methyl-9-oxo-10-oxa-13, 14-disulfide-3, 6-diaza-hexacosyl) azepinediyl) dipropionate; 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol); cKK-E12,3, 6-bis (4- (bis (2-hydroxydodecyl) amino) butyl) piperazine-2, 5-dione; (6 z,9z,28z,31 z) -thirty-seven carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate; hexa (oct-3-yl) 9,9',9",9 '", 9"",9"" ' - (((benzene-1, 3, 5-tricarbonyl) tris (azanediyl)) tris (propane-3, 1-diyl)) tris (azatriyl)) hexanonanoate; heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate; and (((3, 6-dioxopiperazine-2, 5-diyl) bis (butane-4, 1-diyl)) bis (azetidinyl)) tetrakis (ethane-2, 1-diyl) (9Z, 9'Z,9"Z, 9'" Z,12'Z,12"Z, 12'" Z) -tetrakis (octadeca-9, 12-dienoate).
Additionally, in certain embodiments, the lipid nanoparticle may include other lipids. For example, but not limited to, lipid nanoparticles of the presently disclosed subject matter may include phospholipids, cholesterol, polyethylene glycol (PEG) functionalized lipids (PEG-lipids). These lipids may improve certain properties (e.g., stability, biodistribution, etc.) of the lipid nanoparticle. For example, cholesterol enhances the stability of lipid nanoparticles by modulating integrity and rigidity. Non-limiting examples of other lipids present in the lipid nanoparticle include cholesterol, DC-cholesterol, β -sitosterol, BHEM-cholesterol, ALC-0159, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and l, 2-dioleoyl-phosphatidylethanolamine (dos-N-trans-3-PE).
In certain embodiments, the lipid nanoparticle may include a targeting moiety that binds to the ligand. The use of a targeting moiety allows for the selective delivery of an active pharmaceutical ingredient (e.g., a nucleic acid composition as disclosed herein) to a target cell (e.g., a T cell) expressing a ligand. In certain embodiments, the targeting moiety may be an antibody or antigen binding fragment thereof that binds to a cell surface receptor. For example, but not limited to, the targeting domain is an antibody or antigen binding fragment thereof that binds to a receptor expressed on the surface of a T cell (e.g., CD3, CD4, CD8, CD16, CD40L, CD, fasL, CTLA-4, OX40, GITR, LAG3, ICOS, and PD-1).
In certain embodiments, the delivery method is an in vivo delivery method. In certain embodiments, the delivery method is an ex vivo delivery method.
5.6. Polypeptides
The presently disclosed subject matter provides methods for optimizing amino acid sequences or nucleotide sequences by making changes in the sequences. Such alterations may comprise certain mutations, deletions, insertions or post-translational modifications. The presently disclosed subject matter further includes analogs of any of the naturally occurring polypeptides disclosed herein (including but not limited to CD33, CD8, CD28, 4-1BB, and CD3 ζ). Analogs can differ from the naturally occurring polypeptides disclosed herein by amino acid sequence differences, post-translational modifications, or both. Analogs can exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology or identity to all or part of the naturally occurring amino acid sequence of the presently disclosed subject matter. Sequence comparisons are at least 5, 10, 15 or 20 amino acid residues in length, for example at least 25, 50 or 75 amino acid residues, or more than 100 amino acid residues. Also, in an exemplary method of determining the degree of identity, the BLAST program can be used, wherein the probability score between e -3 and e -100 represents closely related sequences. Modification comprises in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modification may occur during polypeptide synthesis or processing, or after treatment with an isolated modifying enzyme. Analogs can also differ from naturally occurring polypeptides by alterations in the primary sequence. These include both natural and induced gene variants (e.g., generated by irradiation or random or site-directed mutagenesis with exposure to ethyl methylsulfonate, such as described in Sambrook, fritsch and Maniatis, molecular Cloning: A LaboratoryManual (2 nd edition), CSH Press,1989 or Ausubel et al, supra). Cyclized peptides, molecules, and analogs containing residues other than an L-amino acid, e.g., a D-amino acid, or a non-naturally occurring or synthetic amino acid, e.g., a beta or gamma amino acid, are also included.
In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any of the polypeptides disclosed herein. As used herein, the term "fragment" means at least 5, 10, 13 or 15 amino acids. In certain embodiments, the fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60 to 80, 100, 200, 300, or more consecutive amino acids. Fragments may be produced by methods known to those of skill in the art or may be produced by normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
5.7. Formulation and administration
The presently disclosed subject matter provides compositions comprising the presently disclosed cells. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The compositions comprising the presently disclosed cells may conveniently be provided in the form of a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, administration of the liquid composition (especially by injection) is somewhat more convenient. On the other hand, the adhesive composition may be formulated within an appropriate viscosity range to provide a longer contact time period with a particular tissue. The liquid or viscous composition may include a carrier, which may be a solvent or dispersion medium containing, for example, water, brine, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.
Sterile injectable solutions may be prepared by incorporating the genetically modified cells in the required amount of the appropriate solvent with various amounts of other ingredients as required. Such compositions may be admixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose and the like. The composition may also be lyophilized. Depending on the route of administration and the desired formulation, the composition may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavouring agents, colouring agents and the like. Standard text (e.g., "REMINGTON' S PHARMACEUTICAL SCIENCE," 17 th edition, 1985) may be queried to prepare a suitable formulation without undue experimentation.
Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial agents as well as antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents which delay absorption, for example, aluminum monostearate (aluminum monostearate) and gelatin. However, any vehicle, diluent or additive used must be compatible with the genetically modified cells in accordance with the presently disclosed subject matter.
The composition may be isotonic, i.e., it may have the same osmotic pressure as blood and tear fluid. The desired isotonicity of the composition may be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride may be particularly suitable for buffers containing sodium ions.
If desired, the viscosity of the composition may be maintained at the selected level using a pharmaceutically acceptable thickener. For example, methylcellulose is readily available and economically viable and readily functional. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomers, and the like. The concentration of the thickener may depend on the agent selected. It is important to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the particular route of administration and the nature of the particular dosage form (e.g., liquid dosage form) (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-filled form).
The compositions comprising the presently disclosed cells may be provided to a subject either systemically or directly for use in treating or ameliorating a disease or disorder. In certain embodiments, the presently disclosed cells or compositions comprising the same are injected directly into an organ of interest (e.g., an organ affected by neoplasia). Alternatively, the presently disclosed cells or compositions comprising the same are provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., tumor vasculature). The expansion and differentiation agents may be provided before, during, or after administration of the cells or compositions to increase production of cells (e.g., T cells or NK cells) in vitro or in vivo.
The presently disclosed cells may be administered in any physiologically acceptable vehicle (typically intravascular), although they may also be introduced into bone or other convenient sites (e.g., thymus) where the cells may find suitable sites for regeneration and differentiation.
The number of cells to be administered may vary for the subject being treated. In certain embodiments, the subject is administered between about 10 4 and about 10 10, between about 10 4 and about 10 7, Between about 10 5 and about 10 7, Between about 10 5 and about 10 9 or between about 10 6 and about 10 8. More potent cells can be administered in even smaller amounts. Typically, at least about l×l0 5 cells will be administered, ultimately reaching about l×l0 10 or more. In certain embodiments, at least about 1×10 5, 5×10 5, 1×10 6, about 5×10 6, and, About 1×10 7, about 5×10 7, about 1×10 8, or about 5×10 8 of the presently disclosed cells. in certain embodiments, about 1 x 10 6 of the presently disclosed cells are administered to a subject. Accurate determination of the dose to be considered an effective dose may be based on individual factors of each subject, including its size, age, sex, weight and condition of the particular subject. Dosages can be readily determined by one of ordinary skill in the art from this disclosure and knowledge in the art.
The presently disclosed cells may include purified cell populations. The percentage of currently disclosed cells in a population can be readily determined by one of skill in the art using a variety of well known methods, such as Fluorescence Activated Cell Sorting (FACS). In populations comprising the presently disclosed immune responsive cells, suitable purities range from about 50% to about 55%, from about 5% to about 60%, and from about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. The dosage may be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). Cells may be introduced by injection, catheters, etc.
The amount of cells and optional additives, vehicles and/or carriers in the composition can be readily determined by one skilled in the art and administered in a method. Generally, any additives (other than the active cells and/or agents) are present in the phosphate buffered saline solution in an amount of about 0.001% to about 50% by weight, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5wt%, about 0.0001 to about 1wt%, about 0.0001 to about 0.05wt%, or about 0.001 to about 20wt%, about 0.01 to about 10wt%, or about 0.05 to about 5wt%. For any composition to be administered to an animal or human, the following may be determined: toxicity, such as by determining Lethal Doses (LD) and LD50 in a suitable animal model (e.g., rodent, such as mouse); the dosage of the composition, the concentration of the components therein, and the timing of administration of the composition that elicits the appropriate response. Such assays do not require undue experimentation based on knowledge of one skilled in the art, the present disclosure, and the literature cited herein. And, the time of sequential administration can be determined without undue experimentation.
In certain embodiments, the composition is a pharmaceutical composition comprising the presently disclosed cells and a pharmaceutically acceptable carrier.
The administration of the composition may be autologous or heterologous. For example, cells can be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived cells or progeny thereof (e.g., in vivo, ex vivo, or in vitro exogenous) may be administered. When the presently disclosed compositions (e.g., pharmaceutical compositions comprising the presently disclosed cells) are administered, the compositions can be formulated into unit-dose injectable forms (solutions, suspensions, emulsions).
The presently disclosed cells and compositions can be administered by any method known in the art, including, but not limited to, oral administration, intravenous administration, subcutaneous administration, intranode administration, intratumoral administration, intrathecal administration, intravitreal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject.
Additionally or alternatively, the presently disclosed subject matter also provides compositions comprising lipid nanoparticles (e.g., described in section 5.5.1) that include a nucleic acid or nucleic acid composition disclosed herein. The compositions comprising the lipid nanoparticles of the present disclosure may conveniently be provided in sterile and/or pyrogen-free form. The composition may be prepared to meet the standards of the United States Pharmacopeia (USP), european Pharmacopeia (EP), british pharmacopeia, and/or international pharmacopeia.
The composition comprising the lipid nanoparticle of the present disclosure may comprise a pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include inert diluents, dispersants, granulating agents, surfactants, emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants and/or oils. In addition, excipients such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavoring and/or perfuming agents may be present in the composition.
In certain embodiments, compositions comprising the presently disclosed lipid nanoparticles can be prepared as injectable formulations. These injectable formulations may include pharmaceutically acceptable vehicles and solvents including, but not limited to, water, ringer's solution, u.s.p., isotonic sodium chloride solution, and/or oil (e.g., oleic acid). In certain embodiments, injectable formulations comprising lipid nanoparticles of the present disclosure may include liquid suspensions of poorly water-soluble crystalline or amorphous materials. The use of these poorly water-soluble materials allows for slow absorption by subcutaneous or intramuscular injection. Alternatively or additionally, compositions comprising the presently disclosed lipid nanoparticles may be prepared for rectal or vaginal administration, oral administration, topical and/or transdermal administration, intradermal administration, pulmonary administration, nasal administration, buccal administration, or ocular administration. Additional information regarding various ways of formulating and preparing pharmaceutical compositions comprising the presently disclosed lipid nanoparticles can be found in Remington THE SCIENCE ANDPRACTICE of Pharmacy, 22 nd edition, a.r. gennaro, lippincott, williams & Wilkins, baltimore, md.,2012.
In certain embodiments, compositions comprising the presently disclosed lipid nanoparticles may be formulated for controlled or sustained release. As used herein, the term "controlled release" refers to a pharmaceutical composition or compound release profile that follows a particular release pattern to achieve a therapeutic result. As used herein, the term "sustained release" refers to a pharmaceutical composition or compound that adheres to the release rate over a particular period of time. The time period may include, but is not limited to, hours, days, weeks, months, and years.
Compositions comprising the presently disclosed lipid nanoparticles may be provided to a subject either systemically or directly for inducing and/or enhancing an immune response against an antigen, and/or treating and/or preventing a tumor, such as a tumor associated with CD 33. In certain embodiments, the presently disclosed lipid nanoparticle or composition comprising the same is provided to an immune responsive cell in vivo. In certain embodiments, the presently disclosed lipid nanoparticle or composition comprising the same is injected directly into an organ of interest (e.g., an organ affected by neoplasia). Alternatively, the presently disclosed lipid nanoparticles or compositions comprising the same are provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., tumor blood vessels). In certain embodiments, the presently disclosed lipid nanoparticle or composition comprising the same is provided ex vivo to an immune response cell. The expansion agent and differentiation agent may be provided before, during, or after administration of the lipid nanoparticle or composition to increase ex vivo or in vivo production of cells (e.g., T cells or NK cells).
The lipid nanoparticles of the present disclosure may be administered in any physiologically acceptable vehicle (typically intravascular), although they may also be introduced into bones or other convenient sites where cells may find suitable sites for regeneration and differentiation (e.g., thymus).
The number of cells to be administered may vary for the subject being treated. In certain embodiments, about 0.001mg/kg to about 10mg/kg, about 0.005mg/kg to about 10mg/kg, about 0.01mg/kg to about 10mg/kg, about 0.05mg/kg to about 10mg/kg, about 0.1mg/kg to about 10mg/kg, about 1mg/kg to about 10mg/kg, about 2mg/kg to about 10mg/kg, about 5mg/kg to about 10mg/kg, about 0.0001mg/kg to about 5mg/kg, about 0.001mg/kg to about 5mg/kg, about 0.005mg/kg to about 5mg/kg, about 0.01mg/kg to about 5mg/kg, about 0.05mg/kg to about 5mg/kg, about 0.1mg/kg to about 1mg/kg, about 2mg/kg to about 5mg/kg, about 2.0001 mg/kg to about 2mg/kg, about 2.0001 mg/kg to about 5mg, about 5mg/kg to about 5mg, about 5mg/kg, about 0.01mg/kg to about 25mg/kg, about 0.01mg/kg to about 5mg/kg, about 5mg/kg to about 5mg/kg, about 0.05mg/kg to about 0.25mg/kg or about 0.1mg/kg to about 0.25mg/kg of the presently disclosed lipid nanoparticle is administered to a subject. In certain embodiments, about 0.005mg/kg to about 2.5mg/kg, about 0.1mg/kg to about 1mg/kg, or about 0.05mg/kg to about 1mg/kg of the presently disclosed cells are administered to a subject. Accurate determination of the dose to be considered an effective dose may be based on individual factors of each subject, including its size, age, sex, weight and condition of the particular subject. Dosages can be readily determined by one of ordinary skill in the art from this disclosure and knowledge in the art. The dosage may be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).
5.8. Therapeutic method
The presently disclosed subject matter provides methods for inducing and/or increasing an immune response in a subject in need thereof. The presently disclosed cells and compositions comprising the same may be used in therapy or medicine. The presently disclosed subject matter provides various methods of using cells (e.g., T cells) or compositions comprising the same. For example, the presently disclosed cells and compositions comprising the same can be used to reduce tumor burden in a subject. The presently disclosed cells can reduce the number of tumor cells, reduce tumor size, and/or eradicate a tumor in a subject. The presently disclosed cells and compositions comprising the same can be used to treat and/or prevent tumors in a subject. The presently disclosed cells and compositions comprising the same may be used to treat or ameliorate a disease or condition in a subject. In certain embodiments, the disease or condition is associated with CD33. In certain embodiments, the disease or condition is associated with overexpression of CD33. In certain embodiments, the disease or condition is a tumor. The presently disclosed cells and compositions comprising the same can be used to extend survival of a subject having a tumor.
Such methods include administering the presently disclosed cells or compositions (e.g., pharmaceutical compositions) comprising the same to achieve a desired effect, e.g., to alleviate an existing condition or prevent recurrence. For the purposes of treatment, the amount administered is an amount effective to produce the desired effect. The effective amount may be provided in one administration or a series of administrations. The effective amount may be provided in bolus or by continuous infusion.
The presently disclosed subject matter provides various methods of using cells (e.g., T cells) or compositions comprising the same. For example, the presently disclosed subject matter provides methods of reducing tumor burden in a subject. In certain embodiments, the method of reducing tumor burden comprises administering to a subject a presently disclosed cell or composition comprising the same. The presently disclosed cells can reduce the number of tumor cells, reduce tumor size, and/or eradicate a tumor in a subject.
The presently disclosed subject matter also provides methods of increasing or extending survival of a subject having a tumor. In certain embodiments, a method of increasing or extending survival of a subject having a tumor or neoplasm comprises administering to the subject the presently disclosed immune responsive cells or composition comprising the same. The method can reduce or eradicate tumor burden in a subject.
In addition, the presently disclosed subject matter provides methods for increasing an immune response in a subject comprising administering to the subject the presently disclosed cells or compositions comprising the same.
The presently disclosed subject matter further provides methods for treating and/or preventing a tumor in a subject comprising administering the presently disclosed cells or compositions comprising the same to the subject.
In certain embodiments, the methods comprise administering to a subject in need thereof the presently disclosed cells or compositions comprising the same. In certain embodiments, the cell is a T cell. The T cells may be CD4 + T cells or CD8 + T cells. In certain embodiments, the T cell is a CD4 + T cell.
In certain embodiments, the tumor is a cancer. In certain embodiments, the cancer is a hematologic cancer or a solid tissue cancer. Non-limiting examples of hematological cancers include Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), myeloproliferative neoplasms (MPN), and chronic myelogenous neoplasms. In certain embodiments, the hematological cancer is AML.
The subject may have advanced forms of the disease, in which case the therapeutic goal may include reducing or reversing disease progression, and/or ameliorating side effects. The subject may have a history of the condition and has been treated, in which case the goal of the treatment generally includes reducing or delaying the risk of relapse.
As a result of the presently disclosed surface expression of antigen recognizing receptor targeted to CD33, adoptively transferred cells (e.g., immune response cells, e.g., T cells or NK cells) are conferred enhanced and selective cytolytic activity at the tumor site. In addition, after they localize to a tumor or viral infection and proliferate, the cells transform the tumor or viral infection site into a highly conductive environment for use by a wide range of immune cells (tumor infiltrating lymphocytes, NK-, NKT-cells, dendritic cells, and macrophages) that are involved in a physiological anti-tumor or antiviral response.
Further modifications may be introduced into CD 33-specific engineered immune cells (e.g., T cells) expressing CD33 to avoid or minimize the risk of immune complications (known as "malignant T cell transformation"), such as graft versus host disease (GvHD). Modification of the engineered immune cell may include engineering a suicide gene into T cells expressing a CD33 specific CAR. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9) and truncated human epidermal growth factor receptor (EGFRt) polypeptides. In certain embodiments, the suicide gene is an EGFRt polypeptide. EGFRt polypeptides may be used to achieve T cell clearance by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt may be covalently linked to the C-terminal end of the intracellular domain of a CD33 specific CAR. Suicide genes may be included in vectors comprising nucleic acids encoding the CD 33-specific CARs of the present disclosure. Integration of suicide genes into the presently disclosed CD 33-specific CARs increases the level of safety, enabling the elimination of most CAR T cells in a very short period of time. The presently disclosed engineered immune cells (e.g., T cells) incorporating suicide genes can be pre-eliminated at a given point in time following CAR T cell infusion, or eradicated at the earliest sign of toxicity.
5.9. Kit for detecting a substance in a sample
The presently disclosed subject matter provides kits for or ameliorating a disease or disorder in a subject, inducing and/or enhancing an immune response in a subject, treating and/or preventing a tumor in a subject, reducing tumor burden in a subject, and/or increasing or extending survival of a subject with a tumor in a subject. In certain embodiments, the kit comprises the presently disclosed cells or compositions comprising the same. In certain embodiments, the kit comprises a sterile container; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable containers known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments. In certain non-limiting embodiments, the kit comprises a nucleic acid molecule encoding the presently disclosed CD 33-targeting antigen recognizing receptor (e.g., CAR).
If desired, the cell and/or nucleic acid molecule is provided along with instructions for administering the cell or nucleic acid molecule to a subject suffering from or at risk of suffering from a disease or disorder (e.g., a tumor). The instructions generally include information regarding the treatment and/or prevention of a tumor using the composition. In certain embodiments, the instructions include at least one of the following: description of therapeutic agents; dosage forms and administration for the treatment or prevention of tumors or neoplasms; notice matters; a warning; indication; contraindications; overdose information; adverse reactions; animal pharmacology; clinical study; and/or references. These instructions may be printed directly on the container (if present), either as a label applied to the container or as a separate sheet, booklet, card or folder provided with or in the container.
5.10. Exemplary embodiments of the invention
A1. In certain non-limiting embodiments, the presently disclosed subject matter provides an antigen recognition receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain specifically binds to CD33, wherein the extracellular antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:
(a) A CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 14 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 23 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 24 or a conservative modification thereof;
(d) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 33 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 34 or a conservative modification thereof;
(e) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39 or a conservative modification thereof;
(f) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 48 or a conservative modification thereof;
(g) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 58 or a conservative modification thereof;
(h) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 67 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68 or a conservative modification thereof;
(i) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 57 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 77 or a conservative modification thereof; or alternatively
(J) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87 or a conservative modification thereof.
A2. the aforementioned antigen recognizing receptor according to A1, wherein the extracellular antigen binding domain is a single chain variable fragment (scFv).
A3. The aforementioned antigen recognizing receptor according to A2, wherein the extracellular antigen binding domain is a human scFv.
A4. The aforementioned antigen recognizing receptor according to A1, wherein the extracellular antigen binding domain is a Fab, which Fab is optionally crosslinked.
A5. The aforementioned antigen recognizing receptor according to A1, wherein the extracellular antigen binding domain is F (ab) 2.
A6. The foregoing antigen recognizing receptor according to any one of A2 to A5, wherein one or more of the scFv, fab and F (ab) 2 is comprised in a fusion protein having a heterologous sequence to form the extracellular antigen binding domain.
A7. The foregoing antigen recognizing receptor according to any one of A1 to A6, wherein the heavy chain variable region comprises:
(a) A CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 2 or a conservative modification thereof, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 3 or a conservative modification thereof, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 4 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 13 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 14 or a conservative modification thereof; or alternatively
(C) A CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 22 or a conservative modification thereof, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 23 or a conservative modification thereof, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 24 or a conservative modification thereof.
A8. the foregoing antigen recognizing receptor according to any one of A1 to A7, wherein the light chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising SEQ ID No. 17 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof;
(d) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 40 or a conservative modification thereof, and a CDR2 comprising SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 42 or a conservative modification thereof;
(e) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51 or a conservative modification thereof;
(f) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61 or a conservative modification thereof;
(g) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71 or a conservative modification thereof;
(h) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80 or a conservative modification thereof; or alternatively
(I) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 42 or a conservative modification thereof.
A9. The foregoing antigen recognizing receptor according to any one of A1 to A8, wherein the light chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising SEQ ID No. 17 or a conservative modification thereof; or alternatively
(C) A CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof.
A10. the aforementioned antigen recognizing receptor according to any one of A1 to A9, wherein:
(a) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 2, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 3, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 4; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 5, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 6, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 7;
(b) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 15, and a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 16, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 17;
(c) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27;
(d) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 32, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 33, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 34; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27;
(e) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 37, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39; the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 40, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 41, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 42;
(f) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 47, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 48; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51;
(g) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 56, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 58; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61;
(h) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 66, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 67, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71;
(i) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 76, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 77; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 79, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80; or alternatively
(J) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 85, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 86, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87; the light chain variable region comprises: CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID NO. 5, CDR2 comprising the amino acid sequence shown in SEQ ID NO. 41, and CDR3 comprising the amino acid sequence shown in SEQ ID NO. 42.
A11. the aforementioned antigen recognizing receptor according to any one of A1 to a10, wherein:
(a) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 2, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 3, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 4; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 5, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 6, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 7;
(b) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 17; or alternatively
(C) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; the light chain variable region comprises: CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID NO:25, CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.
A12. The foregoing antigen recognizing receptor according to any of A1-a 11, wherein the heavy chain variable region comprises an amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO 88.
A13. the antigen recognizing receptor according to any one of A1 to A12, wherein the heavy chain variable region comprises the amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ IDNO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 shown below.
A14. The foregoing antigen recognizing receptor according to any one of A1 to a10, wherein the heavy chain variable region comprises an amino acid sequence shown in: SEQ ID NO. 8, SEQ ID NO. 18 or SEQ ID NO. 28.
A15. The foregoing antigen recognizing receptor according to any one of A1 to a14, wherein the light chain variable region comprises an amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO 92 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous to the amino acid sequences shown below.
A16. the foregoing antigen recognizing receptor according to any one of A1 to a15, wherein the light chain variable region comprises the amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ IDNO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID No. 92 shown below.
A17. The foregoing antigen recognizing receptor according to any one of A1 to a16, wherein the light chain variable region comprises an amino acid sequence shown in: SEQ ID NO. 9, SEQ ID NO. 19 or SEQ ID NO. 29.
A18. the foregoing antigen recognizing receptor according to any one of A1 to a17, wherein:
(a) The heavy chain variable region comprises an amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an amino acid sequence set forth below; and
(B) The light chain variable region comprises an amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO 92 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an amino acid sequence set forth below.
A19. The foregoing antigen recognizing receptor according to any one of A1 to a18, wherein:
(a) The heavy chain variable region comprises the amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 shown below; and
(B) The light chain variable region comprises the amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO 92 shown below.
A20. the aforementioned antigen recognizing receptor according to any one of A1 to a19, wherein:
(a) The heavy chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 8, SEQ ID NO. 18 or SEQ ID NO. 28; and
(B) The light chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 9, SEQ ID NO. 19 or SEQ ID NO. 29.
A21. the foregoing antigen recognizing receptor according to any one of A1 to a20, wherein:
(a) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 9;
(b) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 19;
(c) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29;
(d) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 35 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29;
(e) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 44;
(f) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 52 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 53;
(g) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 63;
(h) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 73;
(i) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 81, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 82;
(j) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 89; or alternatively
(K) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 93.
A22. The antigen recognizing receptor according to any one of A1 to a21, wherein:
(a) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 9;
(b) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 19; or alternatively
(C) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29.
A23. The antigen recognizing receptor according to any one of A1 to a22, wherein the extracellular antigen binding domain comprises a linker between the heavy chain variable region and the light chain variable region.
A24. The aforementioned antigen recognizing receptor according to a23, wherein the linker comprises or consists of an amino acid sequence shown in: 95, 96, 97, 98, 99 or 100.
A25. The foregoing antigen recognizing receptor according to any one of A1 to a24, wherein a signal peptide is covalently attached to the 5' end of the extracellular antigen-binding domain.
A26. The foregoing antigen recognizing receptor according to any one of A1 to a25, wherein the transmembrane domain comprises a CD8 polypeptide, CD28 polypeptide, cd3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, or a combination thereof.
A27. the foregoing antigen recognizing receptor according to any of A1-a 26, wherein the intracellular signaling domain comprises a cd3ζ polypeptide.
A28. the foregoing antigen recognizing receptor according to any of A1-a 27, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region.
A29. the foregoing antigen recognizing receptor according to a28, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.
A30. The antigen recognizing receptor according to any one of A1 to a29, wherein the antigen recognizing receptor is a Chimeric Antigen Receptor (CAR) or a T cell-like fusion protein.
A31. the antigen recognizing receptor according to any one of A1 to a30, wherein the antigen recognizing receptor is a CAR.
A32. the antigen recognizing receptor according to any one of A1 to a31, wherein the antigen recognizing receptor is recombinantly expressed.
A33. the antigen recognizing receptor according to any one of A1 to a32, wherein the antigen recognizing receptor is expressed by a vector.
A34. the antigen recognizing receptor according to A33, wherein the vector is a gamma retrovirus vector.
B1. in certain non-limiting embodiments, the presently disclosed subject matter provides a cell comprising an antigen recognizing receptor according to any one of A1 to a 34.
B2. The aforementioned cell according to B1, wherein the cell is transduced with the antigen recognizing receptor.
B3. the aforementioned cell according to B1 or B2, wherein the antigen recognizing receptor is constitutively expressed on the surface of the cell.
B4. The aforementioned cell according to any one of B1 to B3, wherein the cell is an immune response cell.
B5. the aforementioned cell according to any one of B1 to B4, wherein the cell is a cell of the stranguria line or a cell of the myeloid line.
B6. The aforementioned cell according to any one of B1 to B5, wherein the cell is selected from the group consisting of: t cells, natural Killer (NK) cells, and stem cells from which lymphoid cells may be differentiated.
B7. the aforementioned cell according to any one of B1 to B6, wherein the cell is a T cell.
B8. the aforementioned cell according to B6 or B7, wherein the T cell is a Cytotoxic T Lymphocyte (CTL) or a regulatory T cell.
B9. The aforementioned cell according to B6, wherein the stem cell is a pluripotent stem cell.
B10. The aforementioned cell according to B9, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
C. in certain non-limiting embodiments, the presently disclosed subject matter provides a nucleic acid encoding an antigen recognizing receptor according to any one of A1 to a 34.
D1. In certain non-limiting embodiments, the presently disclosed subject matter provides a vector comprising a nucleic acid according to C.
D2. the foregoing vector according to claim D1, wherein the vector is a gamma retroviral vector.
E1. in certain non-limiting embodiments, the presently disclosed subject matter provides a host cell that expresses a nucleic acid according to C.
E2. the aforementioned host cell according to E1, wherein the host cell is a T cell.
F1. In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising a cell according to any one of B1 to B10.
F2. The aforementioned composition according to F1, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
G. In certain non-limiting embodiments, the presently disclosed subject matter provides a lipid nanoparticle comprising a nucleic acid according to C.
H1. In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising a lipid nanoparticle according to G.
H2. the aforementioned composition according to H1, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
I1. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating or ameliorating a disease or disorder in a subject, the method comprising administering to the subject a cell according to any one of B1 to B10 or a composition according to any one of claims F1, F2, H1, or H2.
I2. the foregoing method according to I1, wherein the disease or condition is a tumor.
J1. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of reducing tumor burden in a subject, the method comprising administering to the subject a cell according to any one of B1 to B10 or a composition according to any one of F1, F2, H1, or H2.
J2. The foregoing method of J1, wherein the method reduces the number of tumor cells, reduces the tumor size, and/or eradicates the tumor in the subject.
K. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating and/or preventing a tumor in a subject, the method comprising administering to the subject a cell according to any one of B1 to B10 or a composition according to any one of F1, F2, H1, or H2.
L1. in certain non-limiting embodiments, the presently disclosed subject matter provides a method of increasing or prolonging survival of a subject having a tumor, the method comprising administering to the subject a cell according to any one of B1 to B10 or a composition according to any one of claims F1, F2, H1, or H2.
L2. the aforementioned method according to L1, wherein the method reduces or eradicates the tumor burden in the subject.
L3. the method according to any one of I2 to L2, wherein the tumor is a cancer.
L4. the method according to any one of I2 to L3, wherein the tumor is a hematological cancer or a solid tissue cancer.
L5. the foregoing method according to any one of I2 to L4, wherein the tumor is selected from the group consisting of: acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), myeloproliferative neoplasms (MPN), and chronic myelogenous neoplasms.
L6. the aforementioned method according to L5, wherein the tumor is Acute Myeloid Leukemia (AML).
L7. the foregoing method according to any one of I2 to L6, wherein the subject is a human.
M1. in certain non-limiting embodiments, the presently disclosed subject matter provides a kit for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and/or preventing a tumor in a subject, and/or increasing or extending survival in a subject with a tumor, the kit comprising a cell according to any one of B1 to B10, a nucleic acid according to C, a lipid nanoparticle according to G, or a composition according to any one of claims F1, F2, H1, or H2.
M2. the aforementioned kit of claim M1, wherein the kit further comprises written instructions for using the cell or composition to treat or ameliorate a disease or disorder in a subject, reduce tumor burden in a subject, treat and/or prevent a tumor in a subject, and/or increase or extend survival of a subject with a tumor.
N. in certain non-limiting embodiments, the presently disclosed subject matter provides a method for producing a CD 33-targeted antigen recognizing receptor according to any one of claims A1 to a34, the method comprising introducing a nucleic acid encoding the antigen recognizing receptor into a cell.
O1. in certain non-limiting embodiments, the presently disclosed subject matter provides a cell according to any one of B1 to B10 or a composition according to F1, F2, H1 or H2 for use in treating or ameliorating a disease.
O2. the aforementioned cells or the aforementioned composition for use according to O1, wherein the disease or condition is a tumor.
O3. the aforementioned cells or the aforementioned composition for use according to O2, wherein the tumor is a cancer.
O4. the foregoing cell or the foregoing composition for use according to any one of O1 to O3, wherein the disease or disorder is selected from the group consisting of: lung neuroendocrine tumors, extrapulmonary neuroendocrine cancers, melanoma, neuroendocrine prostate cancer, breast cancer, gastrointestinal neuroendocrine tumors, pancreatic cancer, medullary thyroid cancer, small cell bladder cancer, ovarian small cell cancer, low grade glioma, glioblastoma, and neuroblastoma.
O5. the foregoing cell or the foregoing composition for use according to O4, wherein the pulmonary neuroendocrine tumor is selected from the group consisting of: lung neuroendocrine cancer, large cell neuroendocrine cancer, and small cell lung cancer.
O6. the aforementioned cells or aforementioned composition for use according to O5, wherein the tumor is small cell lung cancer.
O7. the aforementioned cell or the aforementioned composition for use according to any one of O1 to O6, wherein the subject is a human.
6. Examples
The presently disclosed practice employs, unless otherwise indicated, conventional molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology techniques within the skill of the art. Such techniques are fully explained in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook,1989);"Oligonucleotide Synthesis"(Gait,1984);"Animal Cell Culture"(Freshney,1987);"Methods in Enzymology""Handbook of Experimental Immunology"(Weir,1996);"Gene Transfer Vectors for Mammalian Cells"(Miller and Calos,1987);"CurrentProtocols in Molecular Biology"(Ausubel,1987);"PCR:The Polymerase ChainReaction",(Mullis,1994);"Current Protocols in Immunology"(Coligan,1991)., which are suitable for use in the production of the polynucleotides and polypeptides disclosed herein, and thus may be considered in the preparation and practice of the presently disclosed subject matter. Techniques that are particularly useful for particular embodiments are discussed in the following sections.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use antibodies, multispecific antibodies, compositions comprising the same, screening and treatment methods of the presently disclosed subject matter, and are not intended to limit the scope of what the inventors regard as the presently disclosed subject matter. It is to be understood that various other embodiments may be practiced in light of the general description provided above.
Example 1-design of CAR constructs and cell surface expression
The variable regions of candidate mabs with unique sequences are formatted as scFv. Two antibodies (3P 14 and 4B 2) were specific for IgC2, while the other 5 antibodies (1A 20, 1J19, 1P13, 1P23 and 2N 3) were targeted to IgV domains. These scFv were cloned into a second generation CAR vector containing a CD8 leader, i.e. truncated EGFR (EGFRt), T2A cleavage signal, candidate antibody scFv, myc tag for CAR detection, CD28 transmembrane segment and CD28/CD3 ζ T cell signaling domain flanked by 5 'and 3' Long Terminal Repeats (LTRs) as markers of transduction efficiency, as shown in figure 2. All scFvs were tested in the V L-VH and V H-VL orientations, which were linked by a 15 amino acid glycine serine linker shown in SEQ ID NO:96. The final construct was cloned into retroviral vector SFG 14. Antibody H195 (rituximab) was included as a reference control. The sequence of the lintuzumab variable region is derived from the patent literature.
Retroviral production was performed using packaging cells. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from each donor and activated using standard methods. On days 3 and 4 post-activation, activated T cells were transduced with viral supernatants generated by different CARs. Cells were expanded and tested by flow cytometry using anti-EGFR and anti-Myc antibodies to monitor transduction efficiency and CAR expression, respectively. Overall, each donor with CAR construct achieved transduction efficiencies exceeding 60%. Of the two CARs targeting the CD33 membrane proximal epitope, both 3P14 and 4B2 showed good surface expression only in the V H-VL orientation (see figure 3). The surface expression of 5 CARs targeting the membrane distal epitope varied from low to medium (data not shown).
Example 2-in vitro cytotoxicity of CD33-CAR T cells
The cytolytic capacity of CAR engineered human T cells was assessed by killing assays using U937 or OCi-AML3 AML cell lines with GFP-firefly luciferase reporter. CAR T cells were co-cultured with 1x10 4 target cells in triplicate in 96-well plates at various effector to target ratios. Target cells were plated with non-signaling control CAR T cells of the same cell density to determine maximum luciferase expression as a reference. Bioluminescence was measured after 24 hours and percent lysis was determined as the percent killing observed with nonfunctional CAR T cells.
CARs with antibodies targeting the membrane proximal IgC2 domain of CD33 were assessed on the U937 (CD 33 high) and OCi-AML3 (CD 33 low) cell lines. 3P14 showed killing only in the V H-VL orientation. This is consistent with the lack of surface expression of the scFv in the vL-vH orientation. Surprisingly, however, although vL-vH showed weak surface expression, CARs with 4B2 showed comparable killing in both orientations. Overall, both CARs showed killing activity in excess of 60% compared to the inactive CAR. The killing efficacy was comparable for cells expressing CD33 high (U937) and CD33 low (OCi-AML 3), but slightly different when the E/T ratio was low (see FIGS. 4A and 4B).
Cytotoxicity assessment of CARs with 5 antibodies targeting the CD33 membrane distal IgV domain was performed in U937 of two different donors. One clone (1J 19) showed stronger killing than the reference CAR made with H195 (rituximab) antibody (see fig. 5A and 5B).
Based on these results, two membrane proximal clones 3P14 and 4B2 (designated "TDI-Y-006" and "TDI-Y-007", respectively) with V H-VL scFv orientation and 1J19 were selected for further evaluation.
Example 3 functional characterization of CD33-CAR T cells
To assess the in vitro cytotoxicity of TDI-Y-006 and TDI-Y-007CAR, CAR T cells were co-cultured with a CD33 expressing human AML target line. Three AML cell lines (U937, HL60 and Set 2) were used. Based on the data of example 2, only the V H-VL orientation was selected for further characterization. All experiments included a reference CAR with H195 in V H-VL orientation. When TDI-Y-006 and TDI-Y-007CAR were engineered in T cells from donor 1 (see fig. 6A-6C) or donor 2 (see fig. 6D-6F), various types of AML target cells expressing CD33 were effectively lysed. Overall, more than 80% killing was observed at the highest effector to target ratio. These data are consistent with early evaluation of these clones during the pilot selection phase. When tested using different donors, the 1j19 CAR lyses target cells expressing CD33 equally well (see fig. 7A-7D). All three CAR T cells showed more excellent killing compared to the H195 reference CAR, and clear ordering of potency in TDI-Y-006, TDI-Y-007, 1J19 and H195 order was observed.
Proliferation and cytokine profiles the in vitro T cell proliferation and cytokine activation profiles of TDI-Y-006 and TDI-Y-007CAR T were assessed by co-culturing target cells and effector cells and measuring T cell proliferation and cytokine levels. Briefly, CAR T cells from two different donors were co-cultured with U937 or OCi-AML3 AML cells at a tumor to CAR T cell ratio of 1:5. Every seven days, the remaining tumor and CAR T cells were detected using flow cytometry, and then further stimulated with tumor at a tumor to CAR T cell ratio of 1:5. TDI-Y-006CAR T cells from two donors showed very strong proliferation on U937 cells (CD 33 High height ) compared to H195 reference CAR T, followed by TDI-Y-007 with moderate but still significant stimulation. Similar results were observed for OCi-AML3 cells, with slight differences between the two donors. In all of these settings, H195 reference CAR T cells had only background proliferation levels (see fig. 8A-8D). Taken together, these data demonstrate that TDI-Y-006CAR T, and to a lesser extent TDI-Y-007CAR T, is more effective than H195 reference CAR T cells and exhibits superior proliferation when bound to target cells.
Groups of four cytokine markers (IL-2, GMCSF, IFN-gamma, and TNF-alpha) were evaluated to assess the efficacy of each CAR. In the presence of a 1:1 E:T ratio (40,000 CAR T cells: 40,000U 937 cells) of CD33 expressing U937 target cells, TDI-Y-006CAR T cells from three healthy donors secreted high levels of IL-2, GMCSF, IFN-gamma and TNF-alpha. Figures 9A to 9D show representative data from one donor, while a consistent cytokine profile is seen across all three donors. The level of cytokine secretion induced by TDI-Y-007CAR T cells was 30% to 60% lower than that of TDI-Y-006CAR T cells, depending on the donor. The cytokine secretion level of H195 reference CAR T was similar to TDI-Y-007 or was further reduced, depending on the donor. These data indicate that TDI-Y-006 and TDI-Y-007CAR T induced strong cytokine production after target cell participation. The stronger effect observed for TDI-Y-006 compared to TDI-Y-007 is consistent with its superior proliferative capacity and may be associated with the different affinities of CAR for CD33 on cells.
The production and secretion of these cytokines may enhance the in vitro proliferation of high affinity TDI-Y-006CAR T to a greater extent than low affinity TDI-Y-007CAR T and H195 reference CAR T. Although H195 CAR T has high affinity for the target, cytokine secretion levels are generally lower than the lead CAR, especially TDI-Y-006. This is probably due to the fact that the epitopes of TDI-Y-006 and TDI-Y-007 are located in the membrane proximal IgC2 domain. Binding of the CAR to the CD33 region near the target cell membrane may allow for the establishment of a stronger immune synapse and thus the generation of an effective CAR T. Importantly, IL2 secretion in TDI-Y-006 was consistently higher, followed by TDI-Y-007, indicating proliferation potential in the case of antigen positive cells.
In vivo efficacy: AML xenograft mouse model. The in vivo efficacy of TDI-Y-006 and TDI-Y-007CAR was assessed by Intravenous (IV) injection of U937 tumor cells into NCG mice and infusion of 1 x 10 6 CAR T cells three days later. Mice survival and overall tumor burden were tracked by bioluminescence imaging of tumor cells expressing GFP firefly luciferase. Sustained tumor control was observed by infusion of TDI-Y-006 and TDI-Y-007CAR T cells (see fig. 10A), whereas CAR T with H195 reference antibody only minimally delayed tumor growth compared to non-signaling H195 control. In addition to promoting consistent tumor regression, TDI-Y-006 and TDI-Y-007CAR T cells significantly enhanced survival in tumor-bearing mice compared to H195 reference CAR T (see fig. 10B).
To further verify the in vivo efficacy of the lead CAR T cells, NCG mice were vaccinated with U937 cells and treated with reduced doses of TDI-Y-006 and TDI-Y-007CAR T cells after three days. At a dose of 5×10 5, both CAR T cells showed survival benefits comparable to non-signaling or signaling H195T cells (see fig. 11A and 11B). Interestingly, a finer difference was observed between TDI-Y-006 and TDI-Y-007 when T cells were used at 2.5X10 5, where TDI-Y-006 provided long term survival for mice at this lower CAR T cell dose.
To reproduce these results in tumor models with cells expressing low levels of CD33, NCG mice were IV vaccinated with OCi-AML3 tumor cells and infused with 5 x 10 5 CAR T cells three days later. Mouse survival and overall tumor burden were tracked by bioluminescence imaging of OCi-AML3 tumor cells engineered to express GFP-firefly luciferase. TDI-Y-006 and TDI-Y-007CAR T cells enhanced survival in tumor-bearing mice compared to H195 reference CAR T cells. As seen in the previous experiments, TDI-Y-006CAR T cells showed the strongest tumor control by either cohort averaging (see fig. 12A) or individual animal imaging (see fig. 12B).
To assess in vivo efficacy and ranking of two lead and one backup 1j19 CAR, direct comparisons were made using U937 xenografts. As shown in FIG. 13, a clear ordering in the order TDI-Y-006, TDI-Y-007, 1J19 and H195 can be observed. This is consistent with in vitro killing data and across multiple donors.
In vivo efficacy: patient-derived xenograft mouse model. TDI-Y-006CAR T cells that showed the strongest efficacy in the AML cell line xenograft model described above were further evaluated in an in vivo patient-derived xenograft (PDX) model. NSGS mice were sub-lethally irradiated with 250cGy and PBMC derived from peripheral blast cells of AML patients were infused intravenously. Tumor cells were allowed to grow for 10 days at which time tumor transplantation was verified by flow cytometry staining of human CD45 in peripheral blood. Mice were infused with allogeneic CAR T cells 14 days after tumor inoculation. Seven days after T cell infusion, hcd45+, CD2-, cd33+ AML cells present in bone marrow of TDI-Y-006CART cell treated mice were significantly reduced compared to non-signaling control CAR T cell treated mice (see figure 14).
Furthermore, significant increases in the levels of the pro-inflammatory cytokines IFN- γ and TNF- α were found in the serum of mice treated with TDI-Y-006CAR T cells (see fig. 15A and 15B), indicating that T cell activation and proliferation were also strong in this model.
In vitro hematopoietic stem cell killing ability of CD 33-targeting CAR T cells was assessed using a Colony Forming Unit (CFU) assay. Quantification was performed by total colony count, TDI-Y-006 and TDI-Y-007CAR T cells lysed cd34+ HSCs and were comparable to Gemtuzumab Ozogamicin (GO) used as a positive control (see fig. 16A and 16B). These data indicate that, as with any other CD 33-based therapy, these CAR T cells are likely to affect HSCs, as these cells express CD33. Thus, their clinical use requires rescue of allogeneic stem cell transplantation. Indeed, the ability of TDI-Y-006 and TDI-Y-007 to eliminate HSC can be used to develop a chemotherapy-free transplantation protocol, as these CAR T cells would allow allogeneic cell transplantation without the need for bone marrow preparation of the highly irritating cytotoxic agent. Furthermore, CAR T cells of different doses can be employed to avoid targeting low antigen populations.
Overall, TDI-Y-006 and TDI-Y-007 are novel anti-CD 33 antibodies targeting the proximal domain of the antigen membrane. These targets show excellent in vitro and in vivo efficacy in CAR T format and are superior to existing reference CARs. Disease-related mouse models produce complete eradication of tumors with significant survival benefits. Table 12 below provides a summary of pharmacological properties.
In summary, these novel molecules have great potential as single agents or in combination with other antigens for use in complementary methods for use as next generation AML CAR T therapies.
Example 4-development of Membrane proximal CD33 targeting CAR T cells
This example analyzes the differences between the lintuzumab-CD 28/CD3Z (H195 HLh Z) platform and the currently disclosed antigen recognition receptor targeting the membrane proximal IgC domain of CD33 (CD 33-IgC). This example demonstrates that high affinity CD 33-IgC-targeted CAR T cells have greater in vitro proliferative capacity and increased versatility compared to H195HLh Z, thereby improving survival and tumor control in xenograft models of high and low antigen density and high tumor burden. Surprisingly, low affinity CD33 IgC-targeted CAR T cells exhibited superior functionality compared to high affinity targeting membrane distal H195HLh Z. Overall, these data support targeting CD33-IgC to significantly enhance the efficacy of CAR T cells in treating AML.
Method of
For xenogeneic studies, NOD-Prkdcem26Cd 52Il2rgem26CD22/NjuCrl homologous gene immunodeficiency (NCG) mice were purchased from CHARLES RIVER and subsequently bred and housed in animal facilities under Specific Pathogen Free (SPF) conditions. All experiments used mice of 6 to 12 weeks of age. For CD 33-high tumor studies, 5X 10 4 U937 AML cells expressing Green Fluorescent Protein (GFP) -firefly luciferase were seeded on day 0. For CD 33-low tumor studies, 5X 10 5 OCiAML AML cells expressing GFP-firefly luciferase were seeded on day 0. In both models, mice were treated with various doses of CAR T cells 3 days after tumor inoculation. For patient-derived AML xenograft animal studies, NCG mice were vaccinated with 1x10 6 primary AML blasts and treated with 1x10 6 CAR T cells on day 14.
Two hundred ninety three Glv packaging cells were maintained in high glucose DMEM (dulbeck modified eagle medium) supplemented with 10% heat inactivated FBS (fetal bovine serum) nonessential amino acids (AtlantaBiological Flowery Branch), 2mM L-glutamine (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). The U937 human acute leukemia line, OCiAML human acute myeloid leukemia line, and HL60 human acute myeloid leukemia line were modified to express GFP-firefly luciferase to detect tumors by luminescence in vitro and in vivo. All tumor cell lines were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS nonessential amino acids (Atlanta BiologicalFlowery Branch), 10mM HEPES (hydroxyethylpiperazine ethanesulfonic acid, invitrogen), 2mM L-glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 11mM glucose (Invitrogen). Tumor lines were sorted by Fluorescence Activated Cell Sorting (FACS) based on high expression of GFP. Cell lines are routinely tested for potential mycoplasma contamination.
Selection of scFv Using extracellular Domain of commercially derived or internally produced CD33 recombinant protein by Tri-Institutional Therapeutics DiscoveryInstitute, triI-TDITransgenic mice (Ablexis) were subjected to antibody production and characterization studies. Recombinant human CD33 protein is commercially available from R & D Systems and Acro Biosystems. Mouse and cynomolgus monkey CD33 proteins were purchased from Sino Biological. In addition, the human CD33-IgC domain (residues 140-259) with a terminal mouse IgG1 Fc or 6XHis tag is produced internally. Immunization and screening were performed at LAKEPHARMA (Belmont, CA). Multiple queuesMice were immunized with recombinant protein and one of the cohorts received CD33-IgC. Serum was collected on days 17, 24, 28 and 31 and the immune response was analyzed by enzyme-linked immunosorbent assay (ELISA) using recombinant human full length CD33-6XHis and CD33 IgC2-6XHis proteins. Hybridomas were generated by electrofusion and supernatants were screened by ELISA for recombinant proteins and by flow cytometry for 3T3 cells overexpressing full length CD33 or CD33-IgC alone and AML cell lines with endogenous CD33 expression. Subcloning was performed by limiting dilution and the first 10 lead candidates were sequenced using Ablexis recommended standard IgG primers.
The lead and reference antibodies were recombinantly produced using human IgG1 constant regions. The His-tag recombinant human full-length CD33 or CD33-IgC, mouse full-length CD33 and cynomolgus monkey full-length CD33 protein were used to test selectivity. 5. Mu.g/mL of recombinant protein was captured by pre-blocked Ni-NTA plates. Candidate antibodies were added in triplicate at 10 μg/mL and detected using horseradish peroxidase (HRP) conjugated anti-human Fc antibodies. To assess the binding of CD33 to the cell surface, 3T3 cells overexpressing CD33 and the U937 AML cell line were used. U937 CD33 knockout (CD 33 KO) or NALM6 cells were included as negative controls. NALM6, U937 CD33 + and CD33KO cells were blocked with human IgG Fc on ice for 20 minutes. The recombinant antibodies were then serially diluted starting at a concentration of 100. Mu.g/mL and added on ice for 30 minutes. Alexa Fluor 647 conjugated goat anti-human F (ab') 2 was added to cells on ice for 30 minutes, washed and analyzed by flow cytometry, and normalized to only secondary staining (MFI ratio). EC 50 values were determined by nonlinear regression. Binding affinity and epitope binning to human CD33 protein were measured by Biological Layer Interferometry (BLI) using Octet Red96 e. All experiments were performed using kinetic buffers (PBS pH 7.4,0.01% BSA,0.002% Tween-20). For affinity measurements, antibodies were captured by anti-huFc biosensors and 7-point 2-fold dilution series of huCD33-His was used as analyte. The data were processed by double reference subtraction and the response curve was fitted entirely to the 1:1langmuir binding model.
For epitope binning, human CD33-His was captured by an anti-penta-His biosensor. The 3x3 matrix with 3P14, 4B2A and H195 was tested. A buffer-only reference biosensor was used to determine the overall capture level of each antibody. After pre-binding the antibodies at saturation level, the biosensor was immersed in an antibody solution to assess competition.
Cloning the construct into an SFGgamma retroviral vector having a human signaling domain. The gpg29 fibroblasts (H29) were transiently transfected with the CAR-encoding retroviral construct by generating a retrovirus producer cell line using CaPO4 (Promega) according to the manufacturer's instructions. Supernatant from H29 cells was used to transduce 293Glv to generate a stable retrovirus producer cell line.
T cell isolation and retroviral transduction briefly, peripheral Blood Mononuclear Cells (PBMC) were isolated from a healthy donor peripheral blood or leukocyte bag (New York Blood Center). After lysing the erythrocytes with ACK (potassium ammonium chloride) lysis buffer (Lonza), human T cells were isolated from PBMC (StemCell Technologies) and subsequently activated with 100IU/mL IL-2 and Dynabeads human T activator CD3/CD28 (beads: cell ratio 1:5) (Thermo FisherScientific). Forty-eight hours after initial expansion, T cells were spin seeded with virus supernatant collected from 293Glv9 packaging cells on RetroNectin coated plates (TakaraClontech) for 2 consecutive days. Transduction efficiency was determined by flow cytometry analysis. All experiments were normalized to car+ live cells.
Flow cytometry was performed using a 10-color Gallios B43618 (Beckman Coulter, indianapolis, ind.) and a 14-color Attune NxT (Thermo FISHER SCIENTIFIC) instrument. Data were analyzed using FlowJo (Tree Star). After staining with Phycoerythrin (PE) -conjugated cetuximab antibody and Myc tag (9B11,Alexa Fluor 647,Cell Signaling), transduction efficiency of transduced cells was determined using flow cytometry. DAPI (0.5 mg/mL, SIGMA ALDRICH) or 7-amino-actinomycin D (0.05 mg/mL, bioLegend) and PO-PRO-1 iodine (Invitrogen) staining were used in all experiments to exclude dead cells. The following anti-human antibodies were used for flow cytometry: anti-CD 4 (RPA-T4), anti-CD 8 (SK 1), anti-62L (DREG-56), anti-CD 45RA (HI 100), anti-CD 2 (TS 1/8), anti-CD 371 (50C 1), anti-CD 45 (2D 1), anti-CD 33 (WM 53), and anti-CD 123 (6H 6). The following anti-mouse antibodies were used for flow cytometry: anti-CD 45 (2D 1). All antibodies were purchased from BioLegend, BD Biosciences, invitrogen or eBioscience.
PBMC were isolated from healthy cord blood units (New York Blood Center). After lysing erythrocytes with ACK lysis buffer (Lonza), CD34 + stem cells were isolated and subsequently expanded in serum-free expansion medium (SFEM) II and 1.0 μm UM171 (StemCell Technologies). The expanded CD34 + progenitor cells and CAR T cells were co-cultured at a cell ratio of 1:1 for about 48 hours. Treated human CD34 + progenitor cells were seeded at a density of 3,333 cells/condition in cytokine-supplemented methylcellulose medium (MethoCurt H4435; STEMCELL Technologies) on 6-well plates. Each condition was cultured in triplicate. Colonies were propagated in culture and scored on day 10 and counts averaged across replicates.
The cytolytic capacity of CAR modified human T cells was assessed by a luciferase killing assay. CAR T cells were co-cultured with 1×10 4 target cells, U937-gfpLuc +, or OCiAML3-gfpLuc + tumor cells in variable effector to target ratios in white wall 96-well plates (Thermo Scientific) in quadruplicates in a total volume of 200 μl of cell culture medium. Target cells were plated with non-signaling control CAR T cells of the same cell density to determine maximum luciferase expression (maximum signal) as a reference. Twenty-four hours later, 15 μ g D-fluorescein (Gold Biotechnology) dissolved in 50 μl PBS was added to each well. Luminescence (sample signal) emitted by each sample was detected in a Spark reader (Tecan) and measured using SparkControl software (Tecan). The percent lysis was determined as [100- (sample signal/average maximum signal) ]. Times.100. For the long term killing assay, CAR T cells and tumor cells were co-cultured in duplicate in a white wall 96-well plate (Thermo Scientific) at a ratio of effector cells to tumor cells of 1:40 in a total volume of 200 μl of cell culture medium and imaged using Sartorius IncuCyte S3 over a 138 hour period. Tumor cell killing was measured as total green count over time.
Proliferation assay CAR T cells were co-cultured with U937-gfpLuc + tumor cells or OCiAML-gfpLuc + tumor cells at a CAR T to tumor cell ratio of 1:5. After 7 days, tumors and T cells were detected using flow cytometry after staining with fluorescent-labeled cetuximab. CAR T cells were re-stimulated with fresh tumor cells at the same CAR T: tumor cell ratio of 1:5. After the initial stimulation, the above procedure was repeated weekly for 21 days.
In order to measure in vitro production of T cell cytokines, CAR T cells were co-cultured with antigen positive tumor cells or antigen negative tumor cells at a ratio of 1:1 in 96 well round bottom plates for 24 hours. After 24 hours, the supernatant was collected and analyzed for cytokines on a Luminex IS100 instrument. Cytokines were detected using Luminex FlexMap D systems, luminex xPONENT 4.2 and 12 weight human panel (Millipore).
To measure intracellular T cell cytokine and granzyme B production, CAR T cells were co-cultured at 1:5 with U937-gfpLuc + tumor cells. After about 16 hours, 500X protein transport inhibitor cocktail was added to the cell culture (eBiosciences) and after 6 hours cells were collected, stained with fixable yellow dead cell stain (ThermoFisher Scientific), and fixed and permeabilized with BD Cytofix/Cytoperm permeabilization solution (BD Biosciences) according to the manufacturer's protocol. Cells were counted and stained with the following human antibodies: cetuximab conjugated internally to (phycoerythrin) PE, anti-CD 4 (RPA-T4), anti-CD 8 (SK 1), anti-IL 2 (MQ 1-17H 12), anti-TNF- α (MAb 11), anti-IFN- γ (4 s.b3) and anti-granzyme B (QA 16a 02).
Cells were washed with PBS, resuspended in PBS at a concentration of 3.0X10 6/mL, and incubated for 20 minutes at room temperature in the absence of light with human TruStain FcX Fc receptor blocking solution (BioLegend) and Live/DEAD fixable blue DEAD cell stain (Invitrogen) according to the manufacturer's instructions. The cells were then washed once in a flow-through wash buffer (FWB; RPMI 1640, without phenol red+4% FBS+0.01% sodium azide). The cell and antibody mixtures were incubated in the dark at room temperature for 20 min in the presence of Super Bright staining buffer (eBiosciences) at a staining volume of 100 μl, washed twice in FWB, resuspended in 0.5% paraformaldehyde/PBS, and immediately acquired using a Cytek Aurora 5L flow cytometer (Cytek). Analysis was performed using FlowJo v 10.8.0.
For CD 33-high expression tumor study, NCG mice were vaccinated with 5X 10 4 U937-gfpLuc + tumor cells via the tail vein on day 0. On day 3, mice were blinded randomized to different CAR T cell treatment cohorts, treated with 5×10 4 to 5×10 5 CAR T cells via the tail vein. For CD 33-low expressing tumor studies, NCG mice were vaccinated with 5X 10 5 O CiAML-gfpLuc + tumor cells via the tail vein on day 0. On day 3, mice were blinded randomized to different CAR T cell treatment cohorts, treated with 5×10 5 CAR T cells via the tail vein. U937-gfpLuc + and OCiAML3-gfpLuc + tumor cells all produced very uniform tumor burden and mice were not excluded prior to treatment. Bioluminescence on day 0 was designated 1×10 7. Tumor burden was measured using bioluminescence imaging weekly using the Xenogen IVIS imaging system (Xenogen) and LIVING IMAGE software (Xenogen). For the AML60B patient-derived xenograft model, cryopreservable, viable primary AML specimens were obtained via an MSK study protocol approved by the institutional review board. NCG mice were vaccinated with 1×10 6 cells on day 0. On day 14, mice were randomized to different treatment cohorts and subsequently treated with 1×10 6 CAR T cells via the tail vein. On day 28, bone marrow aspiration was performed and tumor cells were quantified by flow cytometry.
All statistical analyses were performed using GRAPHPAD PRISM software (GraphPad). Data points represent biological replicates and are shown as mean ± Standard Error of Mean (SEM), as shown in the legends. Statistical significance was determined by paired t-test, one-way ANOVA or two-way ANOVA, as shown in the legend. The log rank (Mantel-Cox) test was used to determine the statistical significance of overall survival in the mice survival experiments. Significance with P <0.05; * P <0.01; * P <0.001; and P < 0.0001.
As a result.
CD33-IgC immunization is necessary for the discovery of site-specific binders. In order to generate CD 33-IgC-specific antibodies, mice are immunized with full-length CD33 or CD33-IgC recombinant proteins, since it is not clear whether the distal IgV domain is critical for preserving the IgC conformation required for immune recognition. Hybridomas were generated and 53 hits were identified by ELISA using recombinant human CD 33. The binding on 3T3 cells expressing full length CD33 or CD33-IgC and cells with U937 expressed by endogenous CD33 (AML cell line) was assessed by flow cytometry (fig. 17). The specificity of the conjugates was tested by screening CD33 negative lines (such as U937 with CD33 knockout) using CRISPR/Cas9 or NALM6 (CD 33 negative cell lines). Interestingly, all CD33-IgC domain specific antibodies that showed consistent binding to various cell lines were derived only from the CD33-IgC immune line. From this group, 3 hybridomas were selected and sequenced, thereby identifying 2 unique clones: 3P14 and 4B2A.
Next, the binding specificity and affinity of 3P14, 4B2A and reference CD33 IgV specific monoclonal antibody (mAb) rituximab (H195) were confirmed. Antibodies are recombinantly expressed as a human IgG1 subclass. All mabs bound to full-length human CD33, but not to cynomolgus monkey or mouse orthologs, and as expected, only 3P14 and 4B2A bound to human CD33-IgC (fig. 18A). With Biological Layer Interferometry (BLI), all mabs had similar binding affinity to recombinant full-length CD33, dissociation constant (K D) and binding rate were similar, but the log dissociation rate of H195 was lower compared to 3P14 and 4B2A (fig. 18A). Given that mabs may have different affinities for soluble antigens versus cell-bound antigens, their binding to cell surface CD33 was measured using antigen-overexpressing 3T3 cells (3T 3-CD 33) and the related AML tumor cell line U937 with endogenous expression of CD 33. Affinity scale from highest to lowest was H195>3p14>4B2a, with the most significant differences noted for U937 cells (fig. 18B and 18C).
Next, epitope binning with immobilized CD33 was used to determine if 3P14 and 4B2A bind to similar epitopes and if their different affinities affected the binding of one antibody relative to the other. It was observed that 4B2A failed to demonstrate binding (0%) when 3P14 was the saturated mAb, and that 3P14 was able to bind (68%) when 4B2A was the saturated mAb; no competition for binding was observed between H195 and 3P14 or 4B2A (fig. 18D), indicating that although 3P14 and 4B2A may have overlapping epitopes, both are different from membrane distal H195.
Next, single-chain variable fragments (scFv) of H195, 3P14 and 4B2A were generated in both VH-VL (HL) and VL-VH (LH) orientations and cloned into a bicistronic vector containing a truncated epidermal growth factor receptor tag (EGFRt) and a Myc tag identical to the human CD28/CD3Z (H28Z) signaling motif (FIG. 2). Transduction was assessed positively by EGFR for consistency, which was similar across all constructs (fig. 19). Myc tag staining demonstrated that HL format demonstrated cell surface CAR expression more reliably (fig. 3).
These data show that CD33-IgC immunization is critical for enhancing binding to this domain, and that 3P14 binds to CD33-IgC with higher affinity and possibly recognizes an epitope similar to 4B2A due to the ability of the former to replace the latter during epitope binning. These data also indicate that HL scFv orientation is the preferred format for preclinical testing since it more consistently demonstrates CAR surface expression of our candidate conjugates.
CD 33-IgC-targeted CAR T cells exhibit enhanced functionality in vitro. Low surface antigen density and excessive tumor burden are the primary mechanisms of resistance and treatment failure, leading to impaired tumor control and reduced CAR T cell effector expansion (Majzner et al, cancer discover.10, 702-723 (2020); spiegel et al, nat.med.27,1419-1431 (2021); lock et al, blood Adv 4,4898-4911 (2020)). The CT or TT genotype of rs12459419C > T SNPs is present in about 50% of AML patients, resulting in low CD33 surface expression on malignant cells. Using flow cytometry, the CD33 expression levels on the following 3 cell lines reported with s12459419C > T SNP genotypes were determined: HL60 (CC), U937 (CT) and OCiAML (TT) (Godwin et al, leukemia 35,2496-2507 (2021)). Although HL60 and U937 cell lines expressed the same CD33 expression, in view of the reported genotypes, U937 was chosen to characterize the high-expression CD33 model (U937-CD 33 High height ) and OCiAML3 was chosen to characterize the low-expression CD33 model (OCiAML-CD 33 Low and low ) (fig. 20A). The GFP-firefly luciferase fusion gene (gfpLuc) was used to transduce the target lines to generate U937-CD33 High height gfpLuc+ and OCiAML3-CD33 Low and low gfpLuc+ for in vitro tracking (data not shown).
Next, the ability of CD33-IgC specific CAR T cells to kill tumor cells with high and low CD33 antigen density was determined. In the 24-hour killing assay at various effector to target (E: T) ratios (1:4 to 1:128), at low E: T ratios, 3P14HLh Z and 4B2AHLh Z showed increased dose-dependent killing of U937-CD33 High height compared to H195HLh Z, but at the same low ratio, only 3P14HLh Z showed enhanced cytotoxicity against OCiAML3-CD33 Low and low cells (fig. 20B and 20C). In a 6 day long term kill assay using an E:T ratio of 1:40, 3P14HLh Z and 4B2AHLh Z showed more excellent tumor control capacity for U937-CD33 High height gfpLuc+ compared to H195HLh Z (FIG. 20D), while in the environment of OCiAML3-CD33 Low and low gfpLuc+, 3P14HLh Z showed more excellent kill compared to both 4B2AHLh Z and H195HLh Z (FIG. 20E).
To verify the CD33-IgC specificity of 3P14HLh Z and 4B2AHLh Z, U937-CD33 KOgfpLuc+ cells were transduced with retroviral vectors encoding modified FLAG-tagged CD33-IgC and surface expression confirmed by flow cytometry (FIG. 21A). In contrast to H195HLh Z, both 3P14HLh Z and 4B2AHLh Z lyse target cells in a dose dependent manner (fig. 21B). Importantly, there was no CAR T cell lysis negative control U937-CD33 KOgfpLuc+ (fig. 21C). In view of the reported low levels of CD33 expression on HSCs, colony Forming Unit (CFU) assays were used to determine if 3P14HLh Z and 4B2AHLh Z could detect and eliminate these cells. Complete ablation of colonies was observed with 3P14HLh Z and high dose of Gemtuzumab Ozogamicin (GO) alone (used as positive control), and incomplete ablation of colonies was observed with 4B2AHLh Z or H195HLh Z (fig. 21D).
Given the correlation of CAR T cell expansion with clinical efficacy, the proliferative capacity of 3P14HLh Z and 4B2AHLh Z was tested in the context of recursive stimulation of high and low CD33 antigen density targets. When co-cultured with U937-CD33 High height gfpLuc+ (fig. 20F) and OCiAML-CD 33 Low and low gfpLuc+ cells (fig. 20G), the expansion and persistence of 3P14HLh Z was increased compared to other CAR T cells, although the expansion and persistence amplitude in the latter was inhibited as expected in view of the low antigen density.
Taken together, these data show that high affinity CD33-IgC specific 3P14HLh Z is functionally superior to low affinity CD33-IgC specific 4B2AHLh28Z and high affinity CD33-IgV specific H195HLh Z in high tumor burden environments, as assessed by recursive stimulation or low antigen density encounters. Furthermore, the low affinity membrane proximal 4B2HLh Z exhibited more excellent CAR T activity compared to the high affinity membrane distal H195HLh Z, which underscores the importance of antigen target proximity in the CD33 CAR T cell context.
CAR T cells targeting CD33-IgC with high affinity have versatility. To identify the mechanism by which 3P14HLh Z functionality was enhanced, 24 hour in vitro cytokine production was assessed by co-culturing CAR T cells with U937-CD33 High height gfpLuc+ or OCiAML3-CD33 Low and low gfpLuc+. In comparison to both 4B2AHLh Z and H195HLh Z, 3P14HLh Z secreted elevated levels of Tc1/Th1 cytokine in a target specific manner (fig. 22A). Given that 3P14HLh Z produced higher accumulated cytokines, the role of versatility that has been associated with enhanced clinical responses was determined (Rossi et al, blood 132,804-814 (2018)). After 24 hours of co-culture of CAR T cells with U937-CD33 High height gfpLuc+ (E: T ratio 1:5), intracellular flow cytometry showed increased IFN-. Gamma., IL-2 and granzyme B levels in 3P14HLh Z compared to H195HLh Z (FIG. 22B). In addition, 3P14HLh Z has fewer non-secreting CD4 + cells and more CD4 + and CD8 + cells secreting at least two factors than H195HLh Z. In contrast, 4B2AHLh Z shows a secretory pattern between 3P14HLh Z and H195HLh Z (fig. 22C to 22E).
Given the previous observation of peak CAR T cell proliferation 7 days after U937-CD33 High height gfpLuc+ encountered (fig. 22F), these CAR T cells were further characterized using multiparameter flow cytometry at this time point. Based on CD69 and HLA-DR (DR) positivity, an increase in the number of 3P14HLh Z cells exhibiting an immunophenotype consistent with activation was observed (FIGS. 22F and 22G).
Taken together, these data indicate that 3P14HLh Z cells exhibit a multifunctional soluble factor secretion profile upon antigen encounter and exhibit high levels of activation upon antigen encounter.
CD33-IgC targeted CAR T cells were effective in AML xenograft models. CD 33-directed CAR T cells must exhibit efficacy in the context of low cell number and low antigen density AML. To determine whether 3P14HLh Z and 4B2AHLh Z could control disease in these environments, NCG mice were vaccinated with U937-CD33 High height gfpLuc+ and treated with various low dose CAR T cells (5 x 10 5、2.5×105、1×105 and 5 x 10 4). After 3 days, bioluminescence (BLI) and survival were tracked (fig. 23A). CD 33-IgC-directed CAR T cells exhibited more excellent tumor control and improved survival in a dose-dependent manner compared to H195HLh Z, while 3P14HLh Z exhibited rapid tumor control and prolonged tumor-free status, resulting in improved survival at all dose levels (fig. 23B-23G). The ability of these CAR T cells to control tumor growth in a low antigen density tumor environment was assessed by using the NCG mice implanted OCiAML-CD 33 Low and low gfpLuc+ (figure 23H). In this setting, 3P14HLh Z again showed more excellent tumor control and improved survival compared to 4B2AHLh Z, and 4B2AHLh28Z again showed improved survival compared to H195HLh Z (fig. 23I-23K).
Taken together, these data demonstrate that 3P14HLh Z cells have enhanced functionality in both high and low antigen density xenograft models, allowing long term survival.
CAR T cells targeting CD33-IgC are effective in xenografts derived from AML patients with high tumor burden. To demonstrate transformation correlation, peripheral blast cells from R/R CD33 + AML patients were used to model high tumor burden in vivo (AML 60B; FIG. 24A). AML60B was implanted without conditional irradiation, allowing for increased tumor burden over time and delayed CAR T cell therapy. Fourteen days after NCG mice inoculation, they were randomized and treated with allogeneic H195H28Z, 3P14HLh Z or 4B2ahlh28Z CAR T cells (fig. 24B). Compared to H195HLh Z, 3P14HLh Z and 4B2AHLh Z had more excellent tumor control at day 28 bone marrow aspiration assessment (fig. 24C), with 3P14HLh Z-only treated mice achieving long term survival in most mice (fig. 24D).
Taken together, these data demonstrate that 3P14HLh Z provides excellent tumor control, resulting in long-term survival in vivo in the high tumor burden environment established by R/R primary AML.
Discussion.
Despite the introduction of new agents in AML treatment devices, prognosis of such bone-based malignancies remains very poor; effective interventions such as adoptive T cell therapies are urgently needed. CAR T cells completely alter the treatment of B cell malignancies, but have not shown efficacy against myeloid disorders.
Recently, a phase I trial (NCT 03126864) for treating R/R AML adult patients using autologous CD 33-directed 4-1BB/CD3Z CAR T cells (Tambaro et al, leukemia 35,3282-3286 (2021)) was described. CAR T cell products were successfully manufactured and administered to only 3 out of 10 patients in the panel, in part because of lymphopenia and insufficient numbers of starting T cells obtained in the apheresis step. None of these 3 treated patients observed an anti-leukemia response at the first dose level. This study shows that AML-directed CAR T cells must be able to proliferate and kill robustly at low effector to tumor ratios, as many R/R AML patients suffer from lymphopenia and have a high disease burden of circulating peripheral blast cells. Another recent study describes extensive preclinical screening of CD33 IgV-directed CAR T cells derived from rituximab and gemtuzumab, incorporating CD28/CD3Z or 4-1BB/CD3Z co-stimulatory domains (Qin et al J Immunother Cancer (2021)). This study reported that the rituximab-CD 28/CD3Z platform is the best CAR of the same class for the treatment of AML. The presently disclosed subject matter hypothesizes that the efficacy of CD 33-directed CAR T cells can be improved by targeting the membrane proximal IgC domain of CD33, allowing for more robust T cell functionality and improved tumor control. It was demonstrated that targeting scFv of CD33-IgC with high affinity can significantly increase the potency of CD33 CAR T cells. Surprisingly, the generation of human conjugates with membrane proximal epitopes is dependent on IgC immunization, since full length CD33 immunization always results in the generation of IgV-specific antibodies, which suggests that IgV is an immunodominant epitope. These data indicate that selecting high affinity scFv targeting membrane proximal epitopes via domain-specific immunity can enhance the efficacy of CAR T cells against targets.
Embodiments of the presently disclosed subject matter
From the foregoing description, it will be apparent that variations and modifications of the presently disclosed subject matter may be made to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a list of elements in any definition of a variable herein includes the definition of the variable as any single element or combination (or sub-combination) of listed elements. The recitation of embodiments herein includes the embodiments as any single embodiment or in combination with any other embodiment or portion thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent or publication was specifically and individually indicated to be incorporated by reference.

Claims (76)

1. An antigen recognizing receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain specifically binds to CD33, wherein the extracellular antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID NO.2 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO.3 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 4 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 14 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 24 or a conservative modification thereof;
(d) A CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 32 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 33 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID NO 34 or a conservative modification thereof;
(e) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 37 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39 or a conservative modification thereof;
(f) A CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 47 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 38 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 48 or a conservative modification thereof;
(g) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 56 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID NO. 58 or a conservative modification thereof;
(h) A CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 66 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 67 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68 or a conservative modification thereof;
(i) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 77 or a conservative modification thereof; or alternatively
(J) A CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 86 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87 or a conservative modification thereof.
2. The antigen recognizing receptor according to claim 1, wherein the extracellular antigen-binding domain is a single chain variable fragment (scFv).
3. The antigen recognizing receptor according to claim 2, wherein the extracellular antigen binding domain is a human scFv.
4. The antigen recognizing receptor according to claim 1, wherein the extracellular antigen binding domain is a Fab, which Fab is optionally crosslinked.
5. The antigen recognizing receptor according to claim 1, wherein the extracellular antigen binding domain is F (ab) 2.
6. The antigen recognizing receptor according to any one of claims 2 to 5, wherein one or more of the scFv, fab and F (ab) 2 is comprised in a fusion protein having a heterologous sequence to form the extracellular antigen binding domain.
7. The antigen recognizing receptor according to any one of claims 1 to 6, wherein the heavy chain variable region comprises:
(a) CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2 or a conservative modification thereof,
CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 4 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 14 or a conservative modification thereof; or alternatively
(C) A CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23 or a conservative modification thereof, and
CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24 or a conservative modification thereof.
8. The antigen recognizing receptor according to any one of claims 1 to 7, wherein the light chain variable region comprises:
(a) CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID NO. 5 or a conservative modification thereof,
CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID NO. 6 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 7 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 16 or a conservative modification thereof, and
A CDR3 comprising SEQ ID NO. 17 or a conservative modification thereof;
(c) A CDR1 comprising the amino acid sequence set forth in SEQ ID NO. 25 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO. 26 or a conservative modification thereof, and
A CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof;
(d) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 40 or a conservative modification thereof, and a CDR2 comprising SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 42 or a conservative modification thereof;
(e) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51 or a conservative modification thereof;
(f) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61 or a conservative modification thereof;
(g) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71 or a conservative modification thereof;
(h) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 78 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80 or a conservative modification thereof; or alternatively
(I) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 41 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID No. 42 or a conservative modification thereof.
9. The antigen recognizing receptor according to any one of claims 1 to 8, wherein the light chain variable region comprises:
(a) A CDR1 comprising the amino acid sequence shown in SEQ ID No. 5 or a conservative modification thereof, a CDR2 comprising the amino acid sequence shown in SEQ ID No. 6 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence shown in SEQ ID No. 7 or a conservative modification thereof;
(b) A CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16 or a conservative modification thereof, and a CDR3 comprising SEQ ID No. 17 or a conservative modification thereof; or alternatively
(C) A CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25 or a conservative modification thereof, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26 or a conservative modification thereof, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27 or a conservative modification thereof.
10. The antigen recognizing receptor according to any one of claims 1 to 9, wherein
(A) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 2, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 3, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 4; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 5, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 6, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 7;
(b) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 15, and a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 16, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 17;
(c) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27;
(d) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 32, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 33, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 34; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 25, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 26, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 27;
(e) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 37, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 38, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 39; the light chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 40, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 41, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 42;
(f) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 47, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 38, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 48; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 49, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 50, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 51;
(g) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 56, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 58; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 59, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 60, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 61;
(h) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 66, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 67, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 68; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 69, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 70, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 71;
(i) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 76, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 57, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 77; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 79, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 79, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 80; or alternatively
(J) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 85, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 86, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 87; the light chain variable region comprises: CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID NO. 5, CDR2 comprising the amino acid sequence shown in SEQ ID NO. 41, and CDR3 comprising the amino acid sequence shown in SEQ ID NO. 42.
11. The antigen recognizing receptor according to any one of claims 1 to 10, wherein:
(a) The heavy chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 2, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 3, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 4; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID No. 5, a CDR2, said CDR2 comprising the amino acid sequence shown in SEQ ID No. 6, and a CDR3, said CDR3 comprising the amino acid sequence shown in SEQ ID No. 7;
(b) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 14; the light chain variable region comprises: a CDR1, said CDR1 comprising the amino acid sequence set forth in SEQ ID No. 15, a CDR2, said CDR2 comprising the amino acid sequence set forth in SEQ ID No. 16, and a CDR3, said CDR3 comprising the amino acid sequence set forth in SEQ ID No. 17; or alternatively
(C) The heavy chain variable region comprises: a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24; the light chain variable region comprises: CDR1, said CDR1 comprising the amino acid sequence shown in SEQ ID NO:25, CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.
12. The antigen recognizing receptor according to any one of claims 1 to 11, wherein the heavy chain variable region comprises an amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID NO 88 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO 88.
13. The antigen recognizing receptor according to any one of claims 1 to 12, wherein the heavy chain variable region comprises the amino acid sequence ：SEQ ID NO:8、SEQ ID NO:18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:81 or SEQ ID No. 88 shown in SEQ ID NO.
14. The antigen recognizing receptor according to any one of claims 1 to 10, wherein the heavy chain variable region comprises an amino acid sequence set forth in: SEQ ID NO. 8, SEQ ID NO. 18 or SEQ ID NO. 28.
15. The antigen recognizing receptor according to any one of claims 1 to 14, wherein the light chain variable region comprises an amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID NO 92 that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO 92.
16. The antigen recognizing receptor according to any one of claims 1 to 15, wherein the light chain variable region comprises the amino acid sequence ：SEQ ID NO:9、SEQ ID NO:19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:89 or SEQ ID No. 92 set forth in SEQ ID NO.
17. The antigen recognizing receptor according to any one of claims 1 to 16, wherein the light chain variable region comprises an amino acid sequence set forth in: SEQ ID NO. 9, SEQ ID NO. 19 or SEQ ID NO. 29.
18. The antigen recognizing receptor according to any one of claims 1 to 17, wherein
(A) The heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to an amino acid sequence set forth in seq id no: SEQ ID NO. 8, SEQ ID NO. 18, SEQ ID NO. 28, SEQ ID NO:
35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:
81 or SEQ ID NO. 88; and
(B) The light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to an amino acid sequence set forth in seq id no: SEQ ID NO 9, SEQ ID NO 19, SEQ ID NO 29, SEQ ID NO:
44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:73、SEQ ID NO:82、SEQ ID NO:
89 or SEQ ID NO. 92.
19. The antigen recognizing receptor according to any one of claims 1 to 18, wherein
(A) The heavy chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 8, SEQ ID NO:
18、SEQ ID NO:28、SEQ ID NO:35、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:
62. SEQ ID NO. 72, SEQ ID NO. 81 or SEQ ID NO. 88; and
(B) The light chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO 9, SEQ ID NO:
19、SEQ ID NO:29、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:
73. SEQ ID NO. 82, SEQ ID NO. 89 or SEQ ID NO. 92.
20. The antigen recognizing receptor according to any one of claims 1 to 19, wherein:
(a) The heavy chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 8, SEQ ID NO. 18 or SEQ ID NO. 28; and
(B) The light chain variable region comprises the amino acid sequence set forth in seq id no: SEQ ID NO. 9, SEQ ID NO. 19 or SEQ ID NO. 29.
21. The antigen recognizing receptor according to any one of claims 1 to 20, wherein
(A) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 9;
(b) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 19;
(c) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29;
(d) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 35 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29;
(e) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 44;
(f) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 52 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 53;
(g) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 63;
(h) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 73;
(i) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 81, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 82;
(j) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 89; or alternatively
(K) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 93.
22. The antigen recognizing receptor according to any one of claims 1 to 21, wherein
(A) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 9;
(b) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 19; or alternatively
(C) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 29.
23. The antigen recognizing receptor according to any one of claims 1 to 22, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region.
24. The antigen recognizing receptor according to claim 23, wherein the linker comprises or consists of an amino acid sequence set forth in: SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99 or SEQ ID NO 100.
25. The antigen recognizing receptor according to any one of claims 1 to 24, wherein a signal peptide is covalently attached to the 5' end of the extracellular antigen-binding domain.
26. The antigen recognizing receptor according to any one of claims 1 to 25, wherein the transmembrane domain comprises a CD8 polypeptide, CD28 polypeptide, cd3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, or a combination thereof.
27. The antigen recognizing receptor according to any one of claims 1 to 26, wherein the intracellular signaling domain comprises a cd3ζ polypeptide.
28. The antigen recognizing receptor according to any one of claims 1 to 27, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region.
29. The antigen recognizing receptor according to claim 28, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.
30. The antigen recognizing receptor according to any one of claims 1 to 29, wherein the antigen recognizing receptor is a Chimeric Antigen Receptor (CAR) or a T-cell-like fusion protein.
31. The antigen recognizing receptor according to any one of claims 1 to 30, wherein the antigen recognizing receptor is a CAR.
32. The antigen recognizing receptor according to any one of claims 1 to 31, wherein the antigen recognizing receptor is recombinantly expressed.
33. The antigen recognizing receptor according to any one of claims 1 to 32, wherein the antigen recognizing receptor is expressed by a vector.
34. The antigen recognizing receptor according to claim 33, wherein the vector is a gamma retrovirus vector.
35. A cell comprising the antigen recognizing receptor according to any one of claims 1 to 34.
36. The cell of claim 35, wherein the cell is transduced with the antigen recognizing receptor.
37. The cell of claim 35 or 36, wherein the antigen recognizing receptor is constitutively expressed on the surface of the cell.
38. The cell of any one of claims 35 to 37, wherein the cell is an immune response cell.
39. The cell of any one of claims 35 to 38, wherein the cell is a cell of the stranguria line or a cell of the myeloid line.
40. The cell of any one of claims 35 to 39, wherein the cell is selected from the group consisting of: t cells, natural Killer (NK) cells, and stem cells from which lymphoid cells may be differentiated.
41. The cell of any one of claims 35 to 40, wherein the cell is a T cell.
42. The cell of claim 40 or 41, wherein the T cell is a Cytotoxic T Lymphocyte (CTL) or regulatory T cell.
43. The cell of claim 42, wherein the stem cell is a pluripotent stem cell.
44. The cell of claim 43, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.
45. A nucleic acid encoding the antigen recognizing receptor according to any one of claims 1 to 34.
46. A vector comprising the nucleic acid of claim 45.
47. The vector of claim 46, wherein the vector is a gamma retroviral vector.
48. A host cell expressing the nucleic acid of claim 45.
49. The host cell of claim 48, wherein the host cell is a T cell.
50. A composition comprising a cell according to any one of claims 35 to 44.
51. The composition of claim 50, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
52. A lipid nanoparticle comprising the nucleic acid of claim 45.
53. A composition comprising the lipid nanoparticle of claim 52.
54. The composition of claim 53, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
55. A method of treating or ameliorating a disease or disorder in a subject, the method comprising administering to the subject the cell of any one of claims 35 to 44 or the composition of any one of claims 50, 51, 53 or 54.
56. The method of claim 55, wherein the disease or disorder is a tumor.
57. A method of reducing tumor burden in a subject, the method comprising administering to the subject the cell of any one of claims 35 to 44 or the composition of any one of claims 50, 51, 53, or 54.
58. The method of claim 57, wherein the method reduces the number of tumor cells, reduces tumor size, and/or eradicates a tumor in the subject.
59. A method of treating and/or preventing a tumor in a subject, the method comprising administering to the subject a cell according to any one of claims 35 to 44 or a composition according to any one of claims 50, 51, 53 or 54.
60. A method of increasing or prolonging survival of a subject having a tumor, the method comprising administering to the subject the cell of any one of claims 35 to 44 or the composition of any one of claims 50, 51, 53, or 54.
61. The method of claim 60, wherein the method reduces or eradicates tumor burden in the subject.
62. The method of any one of claims 56-61, wherein the tumor is a cancer.
63. The method of any one of claims 56-62, wherein the tumor is a hematological cancer or a solid tissue cancer.
64. The method of any one of claims 56-63, wherein said tumor is selected from the group consisting of: acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), myeloproliferative neoplasms (MPN), and chronic myelogenous neoplasms.
65. The method of claim 64, wherein the tumor is Acute Myeloid Leukemia (AML).
66. The method of any one of claims 55-65, wherein the subject is a human.
67. A kit for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and/or preventing a tumor in a subject, and/or increasing or prolonging survival of a subject suffering from a tumor, the kit comprising a cell according to any one of claims 35 to 44, a nucleic acid according to claim 45, a lipid nanoparticle according to claim 52, or a composition according to any one of claims 50, 51, 53 or 54.
68. The kit of claim 67, wherein the kit further comprises written instructions for using the cell or composition to treat or ameliorate a disease or disorder in a subject, reduce tumor burden in a subject, treat and/or prevent a tumor in a subject, and/or increase or extend survival of a subject with a tumor.
69. A method for producing a CD 33-targeted antigen recognizing receptor according to any one of claims 1 to 34, the method comprising introducing a nucleic acid encoding the antigen recognizing receptor into a cell.
70. A cell according to any one of claims 35 to 44 or a composition according to any one of claims 50, 51, 53 or 54 for use in treating or ameliorating a disease or disorder in a subject.
71. The cell or composition for use according to claim 70, wherein the disease or disorder is a tumor.
72. The cell or composition for use according to claim 71, wherein the tumor is a cancer.
73. The cell or composition for use according to any one of claims 70 to 72, wherein the disease or condition is selected from the group consisting of: lung neuroendocrine tumors, extrapulmonary neuroendocrine cancers, melanoma, neuroendocrine prostate cancer, breast cancer, gastrointestinal neuroendocrine tumors, pancreatic cancer, medullary thyroid cancer, small cell bladder cancer, ovarian small cell cancer, low grade glioma, glioblastoma, and neuroblastoma.
74. The cell or composition for use according to claim 73, wherein the pulmonary neuroendocrine tumor is selected from the group consisting of: lung neuroendocrine cancer, large cell neuroendocrine cancer, and small cell lung cancer.
75. The cell or composition for use according to claim 74, wherein the tumor is small cell lung cancer.
76. The cell or composition for use according to any one of claims 70 to 75, wherein the subject is a human.
CN202280071579.3A 2021-09-02 2022-09-02 CD 33-targeting antigen recognizing receptor and use thereof Pending CN118369332A (en)

Applications Claiming Priority (4)

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US63/240,196 2021-09-02
US202263306395P 2022-02-03 2022-02-03
US63/306,395 2022-02-03
PCT/US2022/042444 WO2023034560A1 (en) 2021-09-02 2022-09-02 Antigen recognizing receptors targeting cd33 and uses thereof

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