Chimeric antigen receptor targeting sialyl Lewis A and its uses

Abstract

The subject matter of the present disclosure provides methods and compositions for treating cancer (e.g., pancreatic cancer). It relates to antigen recognition receptors (e.g., chimeric antigen receptors (CARs)) that specifically target sialyl Lewis A (e.g., human sialyl Lewis A), and immune response cells comprising such CARs. The sialyl Lewis A-specific CARs of the present disclosure have enhanced immune activation properties, including anti-tumor activity.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 748,198, filed Oct. 19, 2018, the entire contents of which are incorporated herein by reference and for which priority is claimed. FIELD OF THE INVENTION

[0003] The subject matter of the present disclosure provides methods and compositions for treating cancer (e.g., pancreatic cancer). It relates to chimeric antigen receptors (CARs) that specifically target sialyl Lewis A. The subject matter of the present disclosure also provides immune response cells comprising such CARs, and methods of using such CARs and such cells to treat cancer (e.g., pancreatic cancer). BACKGROUND OF THE INVENTION

[0004] Cell-based immunotherapies are therapies with cancer treatment potential. T cells and other immune cells can be modified by introducing genetic material encoding an artificial or synthetic receptor (termed a chimeric antigen receptor (CAR)) specific for a selected antigen to target tumor antigens. Targeted T cell therapies using CARs have shown recent clinical success in treating hematological malignancies (Dunbar et al., Science (2018); 359). To date, responses to CAR therapies targeting solid tumors have been relatively less (Sadelain et al., Nature (2017); 545:423-431). One of the challenges to overcome in all cancers, especially solid tumors, is antigen heterogeneity. Although all or most B cell malignancies express CD19 (Brentjens et al., Nat Med (2003); 9:279-286), many potential CAR targets are expressed in only a subset of all tumor cells in a patient, creating a risk of antigen escape. Low levels of antigen expression may also lead to resistance to CAR therapy (Fry et al., Nat Med (2018); 24:20-28). Targeting two or more antigens can be implemented in the case of an identified escape population or clone (Wilkie et al., J Clin Immunol (2012); 32:1059-1070; Kloss et al., Nat Biotechnol (2013); 31:71-75; Ruella et al., J Clin Invest (2016); 126:3814-3826; Hegde et al., J Clin Invest (2016); 126:3036-3052; Zah et al., Cancer Immunol Res 2016; 4:498-508), but additional approaches are needed to overcome greater or uncertain target heterogeneity. Thus, new treatment strategies are needed to design CARs that target antigens highly expressed in solid tumor cells and strategies that can induce potent anti-cancer effects with minimal toxicity. SUMMARY OF THE INVENTION

[0005] The subject matter of the present disclosure generally provides chimeric antigen receptors (CARs) that target sialyl Lewis A.

[0006] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain cross-competes with a reference antibody or an antigen-binding portion thereof for binding to sialyl Lewis A. In some such embodiments, the reference antibody or an antigen-binding portion thereof that binds to sialyl Lewis A comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising one, two, or three heavy chain complementarity determining regions (CDR1, CDR2, and / or CDR3), and the light chain variable region comprising one, two, or three light chain CDRs (CDR1, CDR2, and / or CDR3), wherein the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 are selected from the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of any one of the antibodies disclosed in U.S. Patent No. 9,475,874, the content of which is incorporated herein by reference in its entirety.

[0007] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain cross-competes with a reference antibody or an antigen-binding portion thereof for binding to sialyl Lewis A, and wherein the reference antibody or an antigen-binding portion thereof comprises: heavy chain variable region CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1; heavy chain variable region CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2; heavy chain variable region CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3; light chain variable region CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4; light chain variable region CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5; and light chain variable region CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6.

[0008] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain binds to the same or overlapping epitopes on sialyl Lewis A as a reference antibody or an antigen-binding portion thereof, and wherein the reference antibody or an antigen-binding portion thereof comprises: heavy chain variable region CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1; heavy chain variable region CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2; heavy chain variable region CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3; light chain variable region CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4; light chain variable region CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5; and light chain variable region CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6.

[0009] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A and comprises: a heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 or a conservative modification thereof, and a light chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 6 or a conservative modification thereof.

[0010] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 2 or a conservative modification thereof, and a light chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 5 or a conservative modification thereof.

[0011] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 1 or a conservative modification thereof, and a light chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof.

[0012] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A and comprises: a heavy chain variable region CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 1; a heavy chain variable region CDR2 that comprises the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain variable region CDR3 that comprises the amino acid sequence shown in SEQ ID NO: 3.

[0013] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A and comprises: a light chain variable region CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 4; a light chain variable region CDR2 that comprises the amino acid sequence shown in SEQ ID NO: 5; and a light chain variable region CDR3 that comprises the amino acid sequence shown in SEQ ID NO: 6.

[0014] In certain embodiments, the extracellular antigen-binding domain comprises: a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0015] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A and comprises a heavy chain variable region having an amino acid sequence that is at least about 80% homologous (e.g., at least about 80% identical) to SEQ ID NO: 7. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7.

[0016] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A and comprises a light chain variable region having an amino acid sequence that is at least about 80% homologous (e.g., at least about 80% identical) to SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8.

[0017] In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A and comprises:

[0018] a) a heavy chain variable region comprising an amino acid sequence that is at least about 80% homologous (e.g., at least about 80% identical) to SEQ ID NO: 7; and

[0019] b) a light chain variable region comprising an amino acid sequence that is at least about 80% homologous (e.g., at least about 80% identical) to SEQ ID NO: 8.

[0020] In certain embodiments, the extracellular antigen-binding domain comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0021] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least about 80% homology (e.g., at least about 80% identity) to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least about 80% homology (e.g., at least about 80% identity) to SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 8.

[0022] In certain embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). In certain embodiments, the extracellular antigen-binding domain comprises a human scFv. In certain embodiments, the extracellular antigen-binding domain comprises an optionally cross-linked Fab. In certain embodiments, the extracellular antigen-binding domain comprises F(ab) 2 . In certain embodiments, one or more of scFV, Fab, and F(ab) 2 are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a signal peptide covalently conjugated to the 5' end of the extracellular antigen-binding domain.

[0023] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof. In certain embodiments, the intracellular domain further comprises at least one co-stimulatory signaling region. In certain embodiments, the at least one co-stimulatory 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 at least one co-stimulatory signaling region comprises a CD28 polypeptide.

[0024] In certain embodiments, the intracellular signaling domain of the CARs described herein comprises a wild-type CD3ζ polypeptide or a modified CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide (a) lacks all or part of at least one or more (e.g., 1, 2, or 3) immunoreceptor tyrosine-based activation motifs (ITAMs), where the ITAMs can be ITAM1, ITAM2, and / or ITAM3 or include ITAM1, ITAM2, and / or ITAM3; and / or (b) lacks all or part of at least one or more (e.g., 1, 2, or 3) basic-rich stretch (BRS) regions, where the BRS regions can be BRS1, BRS2, and BRS3 or include BRS1, BRS2, and BRS3. In certain embodiments, the modified CD3ζ polypeptide included in the intracellular signaling domain of the CARs described herein comprises at least one or more of the following features:

[0025] a) lacks ITAM2 or a portion thereof, optionally further lacking i) ITAM3 or a portion thereof, and / or ii) ITAM1 or a portion thereof;

[0026] b) lacks ITAM1 or a portion thereof, optionally further lacking ITAM3 or a portion thereof;

[0027] c) lacks ITAM3 or a portion thereof;

[0028] d) includes a deletion of ITAM2 or a portion thereof, optionally further including i) a deletion of ITAM3 or a portion thereof, and / or ii) a deletion of ITAM1 or a portion thereof;

[0029] e) includes a deletion of ITAM1 or a portion thereof, optionally further including a deletion of ITAM3 or a portion thereof; and / or

[0030] f) includes a deletion of ITAM3 or a portion thereof.

[0031] In certain embodiments, the modified CD3ζ polypeptide included in the intracellular signaling domain of the CARs described herein comprises at least one or more of the following features:

[0032] a) lacks BRS2 or a portion thereof, and optionally further lacks i) BRS3 or a portion thereof, and / or ii) BRS1 or a portion thereof;

[0033] b) lacks BRS1 or a portion thereof, and optionally further lacks BRS3 or a portion thereof;

[0034] c) lacks BRS3 or a portion thereof; and / or

[0035] d) lacks BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof;

[0036] e) Comprising a deletion of BRS2 or a portion thereof, and optionally further comprising i) a deletion of BRS3 or a portion thereof, and / or ii) a deletion of BRS1 or a portion thereof;

[0037] f) Comprising a deletion of BRS1 or a portion thereof, and optionally further comprising a deletion of BRS3 or a portion thereof;

[0038] g) Comprising a deletion of BRS3 or a portion thereof; and / or

[0039] h) Comprising deletions of BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof.

[0040] In certain embodiments, the modified CD3ζ polypeptide included in the intracellular signaling domain of the CAR described herein lacks ITAM2, ITAM3, BRS2, and BRS3, or comprises deletions of ITAM2, ITAM3, BRS2, and BRS3.

[0041] In certain embodiments, the transmembrane domain of the CAR described herein is or comprises a native or modified transmembrane domain of a molecule selected from: CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, and combinations thereof.

[0042] In certain embodiments, the CAR described herein further comprises, for example, a hinge / spacer region between the extracellular antigen-binding domain and the transmembrane domain of the CAR. In some embodiments, such a hinge / spacer region is or comprises a native or modified hinge / spacer region of a molecule selected from: CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, and combinations thereof.

[0043] In certain embodiments, the CAR described herein comprises a transmembrane domain and a hinge / spacer region, both derived from the same molecule. For example, in certain embodiments, the CAR described herein comprises:

[0044] a) The hinge / spacer region of the CD28 polypeptide and the transmembrane domain of the CD28 polypeptide;

[0045] b) The hinge / spacer region of the CD84 polypeptide and the transmembrane domain of the CD84 polypeptide;

[0046] c) The hinge / spacer region of the CD166 polypeptide and the transmembrane domain of the CD166 polypeptide;

[0047] d) The hinge / spacer region of the CD8a polypeptide and the transmembrane domain of the CD8a polypeptide; or

[0048] e) The hinge / spacer region of the CD8b polypeptide and the transmembrane domain of the CD8b polypeptide.

[0049] In certain embodiments, the CAR comprises the hinge / spacer region of the CD166 polypeptide and the transmembrane domain of the CD166 polypeptide. In certain embodiments, the CARs described herein comprise transmembrane domains and hinge / spacer regions each derived from different molecules. For example, in certain embodiments, such a CAR can comprise the hinge / spacer region of the CD28 polypeptide and the transmembrane domain of the ICOS polypeptide.

[0050] In certain embodiments, the CARs described herein are recombinantly expressed or expressed from a vector. In certain embodiments, such a vector is a retroviral vector (e.g., a gamma-retroviral vector).

[0051] The subject matter of the present disclosure also provides immune response cells comprising a CAR disclosed herein. In certain embodiments, such immune response cells are transduced with a vector comprising a CAR described herein. In certain embodiments, the CARs described herein are constitutively expressed on the surface of immune response cells. Examples of useful immune response cells according to the present disclosure include, but are not limited to, T cells, natural killer (NK) cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursor cells, and pluripotent stem cells (e.g., from which lymphoid cells can be differentiated). In certain embodiments, the immune response cells comprising a CAR described herein are T cells. In certain embodiments, such T cells are selected from cytotoxic T lymphocytes (CTLs), regulatory T cells, and central memory T cells.

[0052] The subject matter of the present disclosure also provides nucleic acid molecules encoding a CAR disclosed herein.

[0053] The subject matter of the present disclosure also provides vectors each comprising a nucleic acid molecule disclosed herein. In certain embodiments, such a vector is a retroviral vector (e.g., a gamma-retroviral vector).

[0054] The subject matter of the present disclosure also provides host cells expressing a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR as disclosed herein. In certain embodiments, such host cells are T cells.

[0055] In addition, the subject matter of the present disclosure provides methods for generating immune response cells that bind to sialyl Lewis A. In certain embodiments, such methods include introducing a nucleic acid sequence encoding a CAR disclosed herein into immune response cells.

[0056] Additionally, the subject matter of the present disclosure provides a composition that includes immune response cells that bind to sialyl Lewis A (e.g., those disclosed herein). In certain embodiments, such a composition is a pharmaceutical composition that includes immune response cells that bind to sialyl Lewis A (e.g., those disclosed herein) and a pharmaceutically acceptable carrier.

[0057] Furthermore, the subject matter of the present disclosure provides methods for treating and / or preventing malignant growth in a subject. In certain embodiments, the method includes administering to the subject an effective amount of the immune response cells disclosed herein or the composition disclosed herein. In certain embodiments, the malignant growth is pancreatic cancer. In certain embodiments, the method reduces or eradicates the tumor burden in the subject. In certain embodiments, the subject is a human.

[0058] The subject matter of the present disclosure also provides a kit for treating and / or preventing malignant growth. In certain embodiments, the kit includes the immune response cells disclosed herein. In certain embodiments, the kit further includes written instructions for using the immune response cells to treat a subject suffering from neoplasia. In certain embodiments, the malignant growth is pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The following detailed description can be understood in conjunction with the accompanying drawings, which are given by way of example and are not intended to limit the invention to the specific embodiments described.

[0060] Figures 1A - 1F Depicts that radiotherapy (RT) sensitizes pancreatic cancer to CAR T cell killing without affecting target antigen expression. Figure 1A Shows tumor cell viability 48 hours after exposure to various doses of radiation. Figure 1B Shows that Capan2 pancreatic cancer cells were exposed to low-dose RT (2 Gy) and incubated with CAR T cells at the indicated ratios for 18 hours 48 hours later, after which the percentage of killing was determined. Figure 1C Shows unchanged target antigen expression levels 48 hours after RT. Figure 1D Shows transcriptome analysis of target cells six hours after RT, revealing many significantly affected apoptotic pathways. Figure 1E Shows the expression and protein levels of TRAIL mRNA in the CAR T cell medium after exposure to target antigen (capan2 cells expressing sialyl Lewis A (Le A )).Figure 1F Shows TRAIL protein quantified in the media of LBBz and L(del)CAR T cells grown on target cells expressing or not expressing the target antigen. LFC = log2 fold change; E:T = effector:target.

[0061] Figures 2A - 2C Depicts that TRAIL expressed by activated CAR T cells has important functions on antigen-negative tumor cells in a heterogeneous tumor population exposed to low-dose radiation. Figure 2A Shows that CAR-activated T cells produce TRAIL, which acts on radiation-sensitized antigen-positive and antigen-negative tumor cells. Figures 2B - 2C Shows that Ag + cells were mixed with luciferase-expressing Ag - cells at a ratio of 75:25, exposed to low-dose RT, co-cultured with the designated CAR T cells for 4 days, and then Ag - cell killing was quantified.

[0062] Figures 3A - 3B Depicts that sensitizing RT transcriptionally primes TRAIL-induced death in pancreatic cancer cells. Figure 3A Shows that in three biological replicates, before and after exposing Capan2 pancreatic cancer cells to RT, RNA expression levels of signaling molecules known to mediate various TRAIL responses (including survival and migration, tumor-supportive inflammation, necroptosis, apoptosis, and death receptor endocytosis) were quantified by RNAseq. Significantly induced and downregulated molecules are shown in red and green, respectively, and their magnitudes are represented by the color gradient. Molecules shown in gray did not change significantly. Figure 3B Shows that two days before co-culturing with unlabeled Ag + cells, annexin-V 595, and TRAIL - / - or TRAIL wt CAR T cells, CTV-labeled Ag - cells were exposed to RT. The cultures were monitored by live video microscopy, and Ag - cell apoptosis was quantified over time.

[0063] Figures 4A - 4M Depicts that sensitizing RT allows CAR T cells to eliminate heterogeneous PDAC in vivo. Figure 4A Shows that Capan2 tumor cells were mixed at 75:25 LeA(+):(-) and then injected into the pancreas of NSG mice. Nine days after tumor formation, the mice were given RT and then CAR T cells. Figure 4B Shows a waterfall plot of the change in tumor volume at death between different treatment groups. Figures 4C - 4HBLI was performed weekly. Figures 4I - 4K showed that T cell infiltration of tumors from CAR- or RT+CAR-treated mice was determined by BLI T cell imaging (detecting G-Luc on transduced T cells) within the first 19 days ( Figure 4I ), and T cell infiltration of tumors from mice sacrificed on day 21 was determined by IHC ( Figures 4J - 4K , all ns). Figure 4L Tumors in progressing mice showed decreased target antigen expression over time as shown by FACS. Figure 4M BLI of mice treated with RT+L(del) or RT+L(del)-TRAIL CAR T cells was depicted.

[0064] Figures 5A - 5G Results of DLBCL patients with heterogeneous tumors treated with palliative RT and CAR T cells were depicted. Figure 5A showed that systemic or local RT was delivered to mice with pancreatic heterogeneous tumors using image-guided radiation, followed by delivery of CAR T cells. Figures 5B - 5D Tumor burden was monitored by BLI. Figures 5E - 5F showed patient biopsies before CAR T cell therapy for CD19 by IHC ( Figure 5E ) and flow cytometry ( Figure 5F ). Figure 5G FDG-PET scans before and 1, 2, and 6 months after palliative leg RT and systemic 1928z CAR T cells were shown.

[0065] Figures 6A - 6C CAR-specific lysis of cells expressing LeA targeting LeA was depicted. Figure 6A LBBz CAR T cell design containing membrane-bound G-Luc for imaging was shown. Figure 6B Endogenous LeA expression on PC3, Capan2, and BxPC3 cells was examined by flow cytometry. Figure 6C showed PC3, Capan2, or BxPC3 cells mixed with LBBz or L28z CAR or untransduced T cells at various effector:target ratios for 18 hours, followed by quantification of target cell killing.

[0066] Figure 7 Capan2 cells were FACS sorted into LeA + and LeA - populations, then mixed at a ratio of 75:25 LeA+ / - tumor cells. LeA - sorted Capan2 cells maintained LeA - .

[0067] Figure 8 depicts TRAIL wt or CRISPR - knocked - out CAR T cells are stimulated on their targets, and then TRAIL mRNA is quantified and shown relative to wt unstimulated CAR T cells.

[0068] Figure 9 depicts the fold mRNA change of molecules known to mediate various TRAIL processes after low - dose RT, including survival and migration, tumor - supportive inflammation, necroptosis, apoptosis, and death receptor endocytosis. Molecules with adjusted p - value < 0.05 are shown.

[0069] Figure 10 depicts a typical T - cell profile after CAR transduction and TCR knockout before in - vivo injection.

[0070] Figure 11 depicts the CAR T - cell tumor infiltration quantified by CTZ T - cell bioluminescence imaging over time, showing that both TRAIL - knocked - out LBBz and L(del)CAR T cells accumulate in pancreatic tumors over time.

[0071] Figures 12A - 12B depicts the in - vivo persistence, tumor penetration, and Ag + tumor cell consumption of CAR T cells in mice bearing heterogeneous Ag+ / - pancreatic cancer. Figure 12A depicts the analysis of the CAR T - cell content of cells isolated from the blood, spleen, and tumors of mice treated with CAR T cells 6 weeks prior (a pure T - cell population control is shown below). Figure 12B Shows IHC of LeA expression from pancreatic tumors at different time points after CAR T - cell treatment, showing the consumption of tumor cells expressing the target antigen throughout the treatment.

[0072] Figure 13 depicts T - cell accumulation in tumors of mice treated with whole - body or local RT. In mice treated with local, whole - body (TBI), or no RT followed by CAR T cells, LBBz CAR T cells in pancreatic tumors are quantified over time using bioluminescence imaging. Detailed Description

[0073] Detailed description of certain exemplary embodiments

[0074] The subject matter of the present disclosure provides antigen - binding proteins that target sialyl Lewis A, such as chimeric antigen receptors (CARs).

[0075] The subject matter of the present disclosure also provides immune response cells (e.g., T cells (e.g., cytotoxic T lymphocytes (CTLs), regulatory T cells, central memory T cells, etc.), natural killer (NK) cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursor cells, and pluripotent stem cells from which lymphoid cells can be differentiated), which include a CAR targeting Lewis A and / or nucleic acids encoding them, and methods of treating and / or preventing tumors (e.g., pancreatic cancer) using such immune response cells.

[0076] I. Certain Definitions

[0077] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of ordinary skill in the art with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings ascribed to them below.

[0078] 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, in accordance with the practice in the art, “about” can mean within 3 or more standard deviations. Alternatively, “about” can mean a range of up to about 20%, preferably up to 10%, more preferably up to 5% and still more preferably up to 1% of a given value. Or, particularly with respect to biological systems or methods, the term can mean within an order of magnitude of the numerical value, preferably within 5-fold and more preferably within 2-fold.

[0079] As used herein, the term "cell population" refers to a group of at least two cells that express similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells that express similar or different phenotypes.

[0080] As used herein, the term "antibody" refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain the ability to bind immunogens. Such fragments are also well known in the art and are frequently used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab') 2 and Fab. F(ab') 2 and Fab fragments that lack the Fc fragment of the intact antibody are cleared from the circulation more rapidly and may have less non-specific tissue binding of the intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). The antibodies of the invention include intact native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V-region fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, the antibody is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain is composed of a heavy-chain variable region (referred to herein simply as V H ) and a heavy-chain constant (C H ) region. The heavy-chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light-chain variable region (referred to herein simply 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 the V L region can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: 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 the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0081] As used interchangeably herein, the terms "antigen-binding portion," "antigen-binding fragment," or "antigen-binding region" of an antibody refer to the region or portion of the antibody that binds to an antigen and confers antigen specificity to the antibody; a fragment of an antigen-binding protein, such as an antibody, includes one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., a peptide / HLA complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding portions encompassed by the term "antibody fragment" of an antibody include: Fab fragment, a monovalent fragment consisting of V L and V H , C L , and CH1 domains; F(ab) 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; Fd fragment consisting of V H and CH1 domains; Fv fragment, which consists of the V L and V H domains of a single arm of an antibody; dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a V H domain; and isolated complementarity-determining regions (CDRs).

[0082] In addition, although the two domains V L and V H of the Fv fragment are encoded by different genes, they can be joined together using recombinant methods by a synthetic linker so that they form a single polypeptide chain, in which the V L and V H regions pair to form a monovalent molecule. These are called single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988, Proc. Natl. Acad. Sci. 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art and are screened for use in the same manner as intact antibodies.

[0083] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy chain (V H ) and light chain (V L ) of an immunoglobulin (e.g., murine or human) that are covalently linked to form a V H ::V L heterodimer. The heavy chain (V H ) and light chain (V L ) are joined directly or via a peptide-encoding linker (e.g., about 10, 15, 20, 25 amino acids), which joins the N-terminus of V H to the VL The C-terminus of or V H The C-terminus of is connected to V L The N-terminus of. The linker is usually rich in glycine to obtain flexibility and rich in serine or threonine to be soluble. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In certain embodiments, the linker comprises an amino acid having the sequence shown in SEQ ID NO: 11 provided below.

[0084]

[0085] In certain embodiments, the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11 is shown in SEQ ID NO: 12 provided below:

[0086]

[0087] Although the constant region is removed and a linker is introduced, the scFv protein still retains the specificity of the original immunoglobulin. The single-chain Fv polypeptide antibody can be prepared by methods including V as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883, 1988). H and V LNucleic acid expression of coding sequences. See also U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Publications Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle Aug. 12, 2012; Shieh et al., J Immunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invest 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(1):31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 2003 278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., Biochim Biophys Acta 2003 1638(3):257-66).

[0088] As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion. For example, digestion of an antibody with papain produces two F(ab) fragments and an Fc fragment (e.g., the heavy (H) chain constant region; the Fc region that does not bind to an antigen).

[0089] As used herein, "F(ab') 2 " refers to an antibody fragment produced by pepsin digestion of a complete IgG antibody, wherein the fragment has two antigen-binding (ab') (bivalent) regions, each of which (ab') region comprises two separate amino acid chains, i.e., a portion of the H chain and a light chain (L) linked by an S-S bond for binding to an antigen, and the remaining portions of the H chains are linked together. The "F(ab') 2 " fragment can be divided into two separate Fab' fragments.

[0090] As used herein, the term "vector" refers to any genetic element that is capable of replicating when combined with an appropriate control element and can transfer a gene sequence into a cell, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. Thus, the term includes cloning and expression vectors, as well as viral vectors and plasmid vectors.

[0091] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence, such as a recombinant DNA molecule, that contains a desired coding sequence and the appropriate nucleic acid sequences necessary for the coding sequence to be expressed operably linked therein. Nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operon (optional), and a ribosome binding site, usually together with other sequences. It is known that eukaryotic cells utilize promoters, enhancers, and termination and polyadenylation signals.

[0092] As used herein, "CDR" is defined as the amino acid sequence of the complementarity-determining regions of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody includes three heavy-chain and three light-chain CDRs or CDR regions in the variable region. The CDRs provide most of the contact residues for the antibody to bind to an antigen or epitope. In certain embodiments, the CDR regions are delineated using the Kabat system (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242).

[0093] As used herein, the term "affinity" refers to a measure of the strength of binding. Without being bound by theory, affinity depends on the tightness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity", which refers to the strength of the antigen-antibody bond after the formation of a reversible complex. Methods for calculating the affinity of an antibody for an antigen are known in the art, including using binding assays to calculate the affinity. The activity of an antibody in a functional assay (e.g., a flow cytometry assay) also reflects the antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using a functional assay (e.g., a flow cytometry assay).

[0094] Nucleic acid molecules useful for the present disclosure include any nucleic acid molecule encoding a polypeptide or a fragment thereof. In certain embodiments, nucleic acid molecules useful for the present disclosure include nucleic acid molecules encoding an antibody or an antigen-binding portion thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. Polynucleotides having "substantial homology" or "substantial identity" to an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing between complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof to form a double-stranded molecule under various stringent conditions. (See, e.g., Wahl, G.M. and S.L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0095] The terms "substantial homology" or "substantial identity" refer to a polypeptide or nucleic acid molecule that exhibits at least 50% homology or identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or even about 99% homologous (e.g., identical) to the sequence used for alignment at the amino acid level or nucleic acid.

[0096] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group, Biotechnology Center, University of Wisconsin, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. In an exemplary method for determining the degree of identity, the BLAST program can be used, where the probability score between e -3 and e -100 indicates closely related sequences.

[0097] In certain embodiments, the terms "cross-compete" or "compete" refer to a reduction or decrease in the binding of the extracellular antigen-binding domain of a CAR of the present disclosure to a given antigen by a reference antibody or an antigen-binding portion thereof (e.g., including the V H and V L CDR1, CDR2, and CDR3 sequences or V H and V LThe situation of binding of a reference antibody or its antigen-binding portion to the same antigen. The terms "cross-competition" or "competition" also refer to the situation where the binding of the reference antibody or its antigen-binding portion to a given antigen reduces or decreases the binding of the extracellular antigen-binding domain of the CARs of the present disclosure to the same antigen. In certain embodiments, a "cross-competitive" or "competitive" extracellular antigen-binding domain binds to the same or substantially the same epitope, overlapping epitope, or adjacent epitope as the reference antibody or its antigen-binding portion.

[0098] As used herein, the term "analogue" refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0099] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, thereby allowing intercellular recognition and / or interaction.

[0100] As used herein, the term "disease" refers to any symptom or disorder that impairs or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia and pathogen infection of cells.

[0101] "Effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical outcome upon treatment. The effective amount can be administered to a subject in one or more doses. For treatment, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse, or slow the progression of a disease (such as neoplasia), or to mitigate the pathological consequences of a disease (such as neoplasia). The effective amount is typically determined by a physician according to the specific circumstances and is within the capabilities of those skilled in the art. When determining the appropriate dose to achieve an effective amount, several factors are usually considered. These factors include the age, sex, and weight of the subject, the disorder being treated, the severity of the disorder, and the form and effective concentration of the immune response cells being administered.

[0102] As used herein, the term "neoplasia" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. The growth of neoplasia is usually uncontrolled and progressive and occurs under conditions that do not cause or result in the cessation of normal cell proliferation. Neoplasia can affect a variety of cell types, tissues, or organs, including but not limited to the organs selected from the following: bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nerve tissue, ovary, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or their tissue or cell types. Neoplasia includes cancers, such as sarcomas, tumors, or plasmacytomas (malignant tumors of plasma cells).

[0103] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample derived from a cell. The nucleic acid can be from another organism or can be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0104] As used herein, the term "immunoresponsive cell" refers to a cell that plays a role in an immune response, or its progenitor or descendant cells.

[0105] As used herein, the term "modulate" refers to a positive or negative change. Exemplary modulations include a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0106] As used herein, the term "increase" refers to a positive change of at least about 5%, including but not limited to a positive change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0107] As used herein, the term "decrease" refers to a negative change of at least about 5%, including but not limited to a negative change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0108] As used herein, the term "isolated cell" refers to a cell that is separated from the molecules and / or cellular components that are naturally associated with the cell.

[0109] As used herein, the terms "isolated," "purified," or "biologically pure" refer to a substance that is, to varying degrees, free of the components that are normally associated with it in its native state. "Isolated" refers to the degree of separation from the original source or environment. "Purified" refers to a degree of separation that is higher than isolated. A "purified" or "biologically pure" protein is sufficiently free of other substances so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the present disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or if it is substantially free of chemical precursors or other chemicals when produced by chemical synthesis. Purity and homogeneity are generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to substantially one band in an electrophoretic gel. For a protein that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins, which can be purified separately.

[0110] As used herein, the term "secreted" means that a polypeptide is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and vesicles that transiently fuse at the cytoplasmic membrane to release the protein extracellularly.

[0111] As used herein, the term "specifically binds" or "binds specifically to" or "specifically targets" refers to a polypeptide or fragment thereof that recognizes and binds to a biomolecule of interest (e.g., a polypeptide), but substantially does not recognize and bind to other molecules in a sample such as a biological sample that includes or expresses human sialyl Lewis A. For example, in some embodiments, when other potential targets are present, the extracellular antigen-binding domain of a CAR described herein interacts with a specific target (e.g., sialyl Lewis A) and is considered to "specifically bind" to the target (e.g., sialyl Lewis A) with which it interacts. In some embodiments, specific binding is evaluated by detecting or determining the degree of association between a target-binding moiety and its ligand; in some embodiments, specific binding is evaluated by detecting or determining the degree of dissociation of a target-binding moiety-ligand complex; in some embodiments, specific binding is evaluated by detecting or determining the ability of a target-binding moiety to compete for the alternative interaction between its ligand and another entity. In some embodiments, specific binding is evaluated by performing such detection or determination within a range of concentrations.

[0112] As used herein, the term "treatment" refers to a clinical intervention that attempts to alter the course of a disease in an individual or cell being treated and can be used to prevent or during the clinical pathologic process. Therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathologic consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remitting or improving the prognosis. By preventing the progression of a disease or disorder, treatment can not only prevent worsening due to the disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but treatment can also prevent the onset of the disorder or the symptoms of the disorder in a subject at risk of having or suspected of having the disorder.

[0113] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc. (e.g., to be the recipient of a particular treatment or from which cells are harvested).

[0114] II. Sialyl Lewis A

[0115] Sialyl Lewis A (also known as Le A , sialyl Le A and SLe A, CAS No. 92448-22-1) is a tetrasaccharide of a sugar sequence including NeuAc(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc. In certain embodiments, Le A comprises the formula:

[0116]

[0117] Le A is present on certain cell surfaces and is involved in the process of cell-cell recognition. It is a surface antigen expressed on tumors such as 75-90% of pancreatic tumors, while its expression on normal human tissues is relatively low.

[0118] III. Chimeric Antigen Receptor (CAR)

[0119] The present disclosure provides chimeric antigen receptors (CARs) that target cancer antigens. In many embodiments, the present disclosure provides CARs that target pancreatic cancer antigens such as sialyl Lewis A.

[0120] A CAR is an engineered receptor that transfers or confers the specificity of interest onto an immune effector cell. CARs can be used to transfer the specificity of monoclonal antibodies onto T cells; the transfer of their coding sequences is facilitated by retroviral vectors.

[0121] There are three generations of CARs in total. "First-generation" CARs generally consist of an extracellular antigen-binding domain (e.g., single-chain variable fragment (scFv)) fused to a transmembrane domain, which is fused to the cytoplasmic / intracellular domain of a T cell receptor chain. "First-generation" CARs generally have the intracellular domain of the CD3ζ chain, which is the main transmitter of signals from the endogenous TCR. "First-generation" CARs can provide de novo antigen recognition and activate CD4 + and CD8 +Both T cells, and is independent of HLA-mediated antigen presentation. "Second-generation" CARs add the intracellular domains from various costimulatory molecules (such as CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail region of the CAR to provide additional signals to the T cells. "Second-generation" CARs include CARs that provide both costimulation (such as CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second-generation" CARs can improve the anti-tumor activity of T cells. For example, in patients with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL), clinical trials targeting the CD19 molecule have demonstrated the powerful efficacy of "second-generation" CAR-modified T cells. "Third-generation" CARs include CARs that provide multiple costimulations (such as CD28 and 4-1BB) and activation (CD3ζ). Those skilled in the art reading this disclosure will recognize that the CAR constructs provided herein can be first-generation, second-generation, or third-generation constructs.

[0122] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CARs of the present disclosure has high binding specificity and high binding affinity for human sialyl Lewis A. For example, in such embodiments, the extracellular antigen-binding domain of the CAR (implemented as, for example, a human scFv or an analogue thereof) binds human sialyl Lewis A with a dissociation constant (K 7 ) of about 2×10 d M or less. In certain embodiments, K d is about 2×10 -7 M or less, about 1×10 -7 M or less, about 5×10 -8 M or less, about 2×10 -8 M or less, about 1×10 -8 M or less, about 9×10 -9 M or less, about 8×10 -9 M or less, about 7×10 -9 M or less, about 6×10 -9 M or less, about 5×10 -9 M or less, about 4×10 -9 M or less, about 3×10 -9 M or less, about 2×10 -9 M or less, or about 1×10 -9 M or less. In certain non-limiting embodiments, K d is about 2×10 - 8 M or less. In certain non-limiting embodiments, K d is about 1×10 -8 M to about 2×10 -8M. In certain non-limiting embodiments, K d is about 1.3×10 -8 M or less. In certain non-limiting embodiments, K d is about 1.8×10 -8 M or less. In certain non-limiting embodiments, K d is about 1×10 -9 M to about 1×10 -8 M.

[0123] Binding of the extracellular antigen-binding domain of the CAR targeting sialyl Lewis A of the present disclosure (implemented as, for example, a human scFv or an analogue thereof) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, biological assay (such as growth inhibition), or Western Blot assay. Each of these assays detects the presence of a specific target protein-antibody complex, typically by employing a labeled reagent (such as an antibody or scFv) specific for the target complex. For example, an scFv can be radiolabeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). The radioisotope can be detected by methods such as using a gamma counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR targeting sialyl Lewis A is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (such as EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (such as ECFP, Cerulean, and CyPet), and yellow fluorescent protein (such as YFP, Citrine, Venus, and YPet). In certain embodiments, the human scFv of the CAR targeting sialyl Lewis A of the present disclosure is labeled with GFP.

[0124] According to the subject matter of the present disclosure, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds sialyl Lewis A (such as human sialyl Lewis A). In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the extracellular antigen-binding domain is an optionally cross-linked Fab. In certain embodiments, the extracellular binding domain is F(ab)2 。In certain embodiments, any of the foregoing molecules can be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a human scFv that specifically binds to human sialyl Lewis A. In certain embodiments, the scFv is identified by screening an scFv phage library.

[0125] Extracellular antigen - binding domain of CAR

[0126] In certain embodiments, the extracellular antigen-binding domain of the CARs described herein comprises a heavy variable region that comprises one, two, or three CDRs (e.g., CDR1, CDR2, and / or CDR3) of an anti-sialyl Lewis A antibody or an antibody-binding fragment thereof as disclosed in U.S. Patent No. 9,475,874 (“the ’874 patent”) (the content of which is incorporated herein by reference in its entirety for the purposes set forth herein). Additionally or alternatively, in certain embodiments, the extracellular antigen-binding domain of the CARs described herein comprises a light variable region that comprises one, two, or three CDRs (e.g., CDR1, CDR2, and / or CDR3) of an anti-sialyl Lewis A antibody or an antibody-binding fragment thereof as disclosed in the ’874 patent (the content of which is incorporated herein by reference in its entirety for the purposes set forth herein). For example, Table 2 of the ’874 patent lists the amino acid and nucleic acid sequences of the CDRs in the heavy and light chains of such anti-sialyl Lewis A antibodies or antibody-binding fragments thereof. Those skilled in the art reading this disclosure will understand that any such sequences can be used in accordance with this disclosure.

[0127] In certain embodiments, the extracellular antigen-binding domain of the CARs described herein comprises (i) the heavy chain variable region of an anti-sialyl Lewis A antibody or an antibody-binding fragment thereof as disclosed in the ’874 patent, and / or (ii) the light chain variable region of an anti-sialyl Lewis A antibody or an antibody-binding fragment thereof as disclosed in the ’874 patent (the content of which is incorporated herein by reference in its entirety for the purposes set forth herein). For example, Figures 1-10 of the ’874 patent list the H V L amino acid sequences. Those skilled in the art reading this disclosure will understand that any such sequences can be used in accordance with this disclosure. Those skilled in the art will also understand that such sequences can be appropriately substituted (e.g., conservatively substituted), deleted, inserted, and / or modified, provided that the resulting sequence is at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more) identical to the corresponding parental sequence and retains the ability to specifically bind sialyl Lewis A.

[0128] In certain embodiments, the extracellular antigen-binding domain (e.g., a human scFv) comprises a heavy-chain variable region that comprises the amino acid sequence shown in SEQ ID NO: 7. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 9. In certain embodiments, the extracellular antigen-binding domain (e.g., a human scFv) comprises a light-chain variable region that comprises the amino acid sequence shown in SEQ ID NO: 8. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8 is shown in SEQ ID NO: 10. The sequences of SEQ ID NOs: 1-10 are described in Table 1 below.

[0129] In certain embodiments, the extracellular antigen-binding domain is a human scFv that specifically binds to sialyl Lewis A (e.g., human sialyl Lewis A), which is designated scFv5B1.

[0130] In certain embodiments, the extracellular antigen-binding domain is a human scFv. In certain embodiments, the extracellular antigen-binding domain comprises a heavy-chain variable region containing the amino acid sequence shown in SEQ ID NO: 7 and a light-chain variable region containing the amino acid sequence shown in SEQ ID NO: 8, optionally having (iii) a linker sequence, such as a linker peptide, between the heavy-chain variable region and the light-chain variable region. In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 11. In certain embodiments, the extracellular antigen-binding domain is a human scFv-Fc fusion protein or a full-length human IgG having V H and V L regions or CDRs selected from Table 1.

[0131] In certain embodiments, the extracellular antigen-binding domain comprises V H which V H comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology (e.g., identity) to the amino acid sequence shown in SEQ ID NO: 7 as shown in Table 1. For example, the extracellular antigen-binding domain comprises V H which V H comprises an amino acid sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology (e.g., identity) to the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the extracellular antigen-binding domain comprises V H, which comprises the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the extracellular antigen-binding domain comprises V L , and this V L comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology (e.g., identity) to the amino acid sequence shown in SEQ ID NO: 8 as shown in Table 1. For example, the extracellular antigen-binding domain comprises V L , and this V L comprises an amino acid sequence having 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% homology (e.g., identity) to the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises V L , which comprises the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises V H and V L , and this V H comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology (e.g., identity) to the amino acid sequence shown in SEQ ID NO: 7, and this V L comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology (e.g., identity) to the amino acid sequence shown in SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises V H containing the amino acid sequence shown in SEQ ID NO: 7 and V L containing the amino acid sequence shown in SEQ ID NO: 8.

[0132] In certain embodiments, the extracellular antigen-binding domain of the CAR described herein comprises at least one or more (e.g., 1, 2, or 3) heavy chain variable regions (V H ) CDRs that specifically target sialyl Lewis A. For example, in some embodiments, the extracellular antigen-binding domain of the CAR described herein comprises at least one or more (e.g., 1, 2, or 3) of the following: (i) V H CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or its conservative modification, (ii) V HCDR2, and (iii) a V containing the amino acid sequence shown in SEQ ID NO: 3 or a conservative modification thereof H CDR3, as shown in Table 1. In certain embodiments, the extracellular antigen-binding domain comprises a V containing the amino acid sequence shown in SEQ ID NO: 1 or a conservative modification thereof H CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 2 or a conservative modification thereof H CDR2, and a V containing the amino acid sequence shown in SEQ ID NO: 3 or a conservative modification thereof H CDR3, as shown in Table 1. In certain embodiments, the extracellular antigen-binding domain comprises a V containing the amino acid sequence shown in SEQ ID NO: 1 H CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 2 H CDR2 and a V containing the amino acid sequence shown in SEQ ID NO: 3 H CDR3.

[0133] In certain embodiments, the extracellular antigen-binding domain of the CAR described herein comprises at least one or more (e.g., 1, 2, or 3) light chain variable regions (V) that specifically target sialyl Lewis A L ) CDRs. For example, in some embodiments, the extracellular antigen-binding domain of the CAR described herein comprises at least one or more (e.g., 1, 2, or 3) of the following: (i) a V L CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof, (ii) a V L CDR2 that comprises the amino acid sequence shown in SEQ ID NO: 5 or a conservative modification thereof, and (iii) a V L CDR3 that comprises the amino acid sequence shown in SEQ ID NO: 6 or a conservative modification thereof, as shown in Table 1. In certain embodiments, the extracellular antigen-binding domain comprises a V containing the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof L CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 5 or a conservative modification thereof L CDR2 and a V containing the amino acid sequence shown in SEQ ID NO: 6 or a conservative modification thereof L CDR3, as shown in Table 1. In certain embodiments, the extracellular antigen-binding domain comprises a V containing the amino acid sequence shown in SEQ ID NO: 4 L CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 5 LCDR2 and V containing the amino acid sequence shown in SEQ ID NO: 6 L CDR3.

[0134] In certain embodiments, the extracellular antigen-binding domain of the CARs described herein includes a V containing the amino acid sequence shown in SEQ ID NO: 1 or a conservative modification thereof H CDR1 and a V containing the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof L CDR1. In certain embodiments, the extracellular antigen-binding domain of the CARs described herein includes a V containing the amino acid sequence shown in SEQ ID NO: 2 or a conservative modification thereof H CDR2 and a V containing the amino acid sequence shown in SEQ ID NO: 5 or a conservative modification thereof L CDR2. In certain embodiments, the extracellular antigen-binding domain of the CARs described herein includes a V containing the amino acid sequence shown in SEQ ID NO: 3 or a conservative modification thereof H CDR3 and a V containing the amino acid sequence shown in SEQ ID NO: 6 or a conservative modification thereof L CDR3.

[0135] In certain embodiments, the extracellular antigen-binding domain of the CARs described herein includes a V containing the amino acid sequence shown in SEQ ID NO: 1 or a conservative modification thereof H CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 2 or a conservative modification thereof H CDR2, a V containing the amino acid sequence shown in SEQ ID NO: 3 or a conservative modification thereof H CDR3, a V containing the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof L CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 5 or a conservative modification thereof L CDR2 and a V containing the amino acid sequence shown in SEQ ID NO: 6 or a conservative modification thereof L CDR3.

[0136] In certain embodiments, the extracellular antigen-binding domain includes a V containing the amino acid sequence shown in SEQ ID NO: 1 H CDR1, a V containing the amino acid sequence shown in SEQ ID NO: 2 H CDR2, a V containing the amino acid sequence shown in SEQ ID NO: 3 H CDR3, a V containing the amino acid sequence shown in SEQ ID NO: 4 LCDR1, V containing the amino acid sequence shown in SEQ ID NO: 5 L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 6 L CDR3.

[0137] In certain embodiments, the CDRs are provided as follows (e.g., according to Kabat numbering).

[0138] Table 1

[0139]

[0140] As used herein, the terms "conservative modification" or "conservative sequence modification" refer to amino acid modifications that do not significantly affect or alter the binding characteristics (e.g., specificity and / or affinity) of the CARs of the present disclosure, including amino acid sequences (e.g., the extracellular antigen-binding domain of the CAR). Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of the CARs of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified 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. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within the CDR regions can be replaced with other amino acid residues from the same group, and the retained function of the antibody that has been altered can be tested using the functional assays described herein (i.e., the functions listed in (c) to (l) above). 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 the specified sequence or CDR regions are altered.

[0141] For example, in some embodiments, the V included in the CARs described herein H and / or V LConservative modifications of an amino acid sequence (e.g., SEQ ID NOs: 1-10 shown in Table 1) are amino acid sequences that have 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 the specified sequence, contain at least one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) substitutions (e.g., conservative substitutions), insertions, and / or deletions relative to the specified sequence, but retain the ability to bind sialyl Lewis A (e.g., human sialyl Lewis A). In some embodiments, the V included in the CARs described herein H and / or V L such conservative modifications of the amino acid sequence (e.g., SEQ ID NOs: 1-10 shown in Table 1) retain at least 70% or more, including for example at least 80%, at least 90%, at least 95% or more and up to 100% of the binding affinity of the corresponding unmodified V H and / or V L amino acid sequence for sialyl-Lewis A. For example, in certain embodiments, the extracellular antigen-binding domain binds sialyl Lewis A (e.g., human sialyl Lewis A) with a binding affinity (K -8 ) of about 3×10 d or less. In certain embodiments, the extracellular antigen-binding domain binds sialyl Lewis A (e.g., human sialyl Lewis A) with a binding affinity (K -8 ) of about 2×10 d or less. In certain embodiments, the extracellular antigen-binding domain binds sialyl Lewis A (e.g., human sialyl Lewis A) with a binding affinity (K -8 ) of about 1.3×10 d or less. In certain embodiments, the extracellular antigen-binding domain binds sialyl Lewis A (e.g., human sialyl Lewis A) with a binding affinity (K -8 ) of about 1.8×10 d or less. In certain embodiments, the extracellular antigen-binding domain binds sialyl Lewis A (e.g., human sialyl Lewis A) with a binding affinity (K -9 ) of about 1×10 -7 to about 1×10 d . In certain embodiments, the extracellular antigen-binding domain binds sialyl Lewis A (e.g., human sialyl Lewis A) with a binding affinity (K -8to about 2×10 -8 binding affinity (K d ) binds to sialyl Lewis A, such as human sialyl Lewis A. In certain embodiments, 1 to 10 amino acids in total in SEQ ID NO: 7 or 8 are substituted, inserted, and / or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the CDRs of the extracellular antigen-binding domain (e.g., in the FRs). One of ordinary skill in the art reading Table 1 provided herein will be able to identify and determine the amino acid and / or nucleic acid sequences of the framework regions (FRs) based on the sequence information provided. In certain embodiments, the extracellular antigen-binding domain comprises a V H and / or V L sequence selected from SEQ ID NO: 7 and 8 (including post-translational modifications of the sequence (SEQ ID NO: 7 or 8)).

[0142] As used herein, the percent homology between two amino acid sequences is equivalent 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 = number of identical positions # / total number of positions # × 100), where consideration is given to the number of gaps and the length of each gap, which are introduced to achieve the best alignment of the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using mathematical algorithms.

[0143] 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, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available from www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0144] Additionally or alternatively, the amino acid sequences of the subject matter of the present disclosure can further be used as a "query sequence" to search public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. The BLASTP program can be used to perform BLAST protein searches with a score = 50 and word length = 3 to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0145] In certain embodiments, the extracellular antigen-binding domain of the CARs of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising, for example, the V H CDR1, CDR2, and CDR3 sequences of any one of the scFvs of the present disclosure and the V L CDR1, CDR2, and CDR3 sequences to bind to sialyl Lewis A (e.g., human sialyl Lewis A). In certain embodiments, the extracellular antigen-binding domain of the CARs of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising the V H and V L sequences to bind to sialyl Lewis A (e.g., human sialyl Lewis A).

[0146] In certain embodiments, the extracellular antigen-binding domain of the CARs of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising the V H CDR1, CDR2, and CDR3 sequences of scFv 5B1 and the V L CDR1, CDR2, and CDR3 sequences to bind to sialyl Lewis A (e.g., human sialyl Lewis A). For example, the extracellular antigen-binding domain of the CARs of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising the V H CDR1 containing the amino acid sequence shown in SEQ ID NO: 1; the V H CDR2 containing the amino acid sequence shown in SEQ ID NO: 2; the V H CDR3 containing the amino acid sequence shown in SEQ ID NO: 3; the V containing the amino acid sequence shown in SEQ ID NO: 4L CDR1; a V containing the amino acid sequence shown in SEQ ID NO: 5 L CDR2; and a V containing the amino acid sequence shown in SEQ ID NO: 6 L The extracellular antigen-binding domain of the CAR of the present disclosure cross-competes with a reference antibody or an antigen-binding portion thereof containing CDR3 to bind to sialyl Lewis A (such as human sialyl Lewis A). In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure cross-competes with a reference antibody or an antigen-binding portion thereof comprising the V H and V L sequences to bind to sialyl Lewis A. For example, the extracellular antigen-binding domain of the CAR of the present disclosure cross-competes with a reference antibody or an antigen-binding portion thereof comprising a V containing the amino acid sequence shown in SEQ ID NO: 7 H and a V containing the amino acid sequence shown in SEQ ID NO: 8 L to bind to sialyl Lewis A.

[0147] In certain embodiments, the extracellular antigen-binding domain binds to the same or overlapping epitopes on sialyl Lewis A (such as human sialyl Lewis A) as the reference antibody or an antigen-binding portion thereof. For example, the extracellular antigen-binding domain of the CAR of the present disclosure binds to the same or overlapping epitopes on sialyl Lewis A (such as human sialyl Lewis A) as a reference antibody or an antigen-binding portion thereof comprising, for example, the V H CDR1, CDR2, and CDR3 sequences and V L CDR1, CDR2, and CDR3 sequences. In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure binds to the same or overlapping epitopes on sialyl Lewis A (such as human sialyl Lewis A) as a reference antibody or an antigen-binding portion thereof comprising, for example, the V H and V L sequences.

[0148] In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure binds to the same or overlapping epitopes on sialyl Lewis A (such as human sialyl Lewis A) as a reference antibody or an antigen-binding portion thereof comprising the V H CDR1, CDR2, and CDR3 sequences and V L CDR1, CDR2, and CDR3 sequences. For example, the extracellular antigen-binding domain of the CAR of the present disclosure binds to the same or overlapping epitopes on sialyl Lewis A (such as human sialyl Lewis A) as a reference antibody or an antigen-binding portion thereof comprising a V containing the amino acid sequence shown in SEQ ID NO: 1 H CDR1; a V containing the amino acid sequence shown in SEQ ID NO: 2 HCDR2; V containing the amino acid sequence shown in SEQ ID NO: 3 H CDR3; V containing the amino acid sequence shown in SEQ ID NO: 4 L CDR1; V containing the amino acid sequence shown in SEQ ID NO: 5 L CDR2; and V containing the amino acid sequence shown in SEQ ID NO: 6 L The reference antibody or antigen-binding portion thereof having CDR3 binds to the same or overlapping epitopes on sialyl Lewis A (e.g., human sialyl Lewis A). In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure binds to the same or substantially the same epitopes on sialyl Lewis A (e.g., human sialyl Lewis A) as the reference antibody or antigen-binding portion thereof comprising the V of scFv 5B1 H and V L sequence. For example, the extracellular antigen-binding domain of the CAR of the present disclosure binds to the same or overlapping epitopes on sialyl Lewis A (e.g., human sialyl Lewis A) as the reference antibody or antigen-binding portion thereof comprising the V containing the amino acid sequence shown in SEQ ID NO: 7 H and the V containing the amino acid sequence shown in SEQ ID NO: 8 L of the reference antibody or antigen-binding portion thereof.

[0149] Extracellular antigen-binding domains that cross-compete or compete with a reference antibody or its antigen-binding portion for binding to sialyl Lewis A (e.g., human sialyl Lewis A) can be identified by using conventional methods known in the art, including but not limited to ELISA, radioimmunoassay (RIA), Biacore, flow cytometry, Western blotting, and any other suitable quantitative or qualitative antibody-binding assay. Competitive ELISA is described in Morris's "Epitope Mapping of Protein Antigens by Competition ELISA" The Protein Protocols Handbook (1996), pp 595-600, edited by J. Walker (which is incorporated herein by reference in its entirety). In certain embodiments, the antibody-binding assay includes measuring the initial binding of the reference antibody to sialyl Lewis A, mixing the reference antibody with the tested extracellular antigen-binding domain, measuring the second binding of the reference antibody to sialyl Lewis A in the presence of the tested extracellular antigen-binding domain, and comparing the initial binding of the reference antibody with the second binding, wherein a decrease in the second binding of the reference antibody to sialyl Lewis A compared to the initial binding indicates that the tested extracellular antigen-binding domain cross-competes with the reference antibody for binding to sialyl Lewis A, e.g., recognizes the same or substantially the same epitope, overlapping epitopes, or adjacent epitopes. In certain embodiments, the reference antibody is labeled, for example, with a fluorescent dye, biotin, or peroxidase. In certain embodiments, sialyl Lewis A is expressed in cells, e.g., in a flow cytometry assay. In certain embodiments, sialyl Lewis A is immobilized on a surface including a Biacore ship (e.g., in a Biacore assay) or other medium suitable for surface plasmon resonance analysis. The binding of the reference antibody in the presence of a completely unrelated antibody (that does not bind to sialyl Lewis A) can serve as a control high value. A control low value can be obtained by incubating the labeled reference antibody with an unlabeled reference antibody, where the labeled reference antibody competes and shows reduced binding. In certain embodiments, a tested extracellular antigen-binding domain that reduces the binding of the reference antibody to sialyl Lewis A 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% is considered an extracellular antigen-binding domain that cross-competes with the reference antibody for binding to sialyl Lewis A. In certain embodiments, the assay is performed at room temperature.

[0150] In certain embodiments, the antibody binding assay comprises measuring an initial binding of a tested extracellular antigen binding domain to sialyl Lewis A, mixing the tested extracellular antigen binding domain with a reference antibody, measuring a second binding of the tested extracellular antigen binding domain to a sialyl Lewis A polypeptide in the presence of the reference antibody, and comparing the initial binding of the tested extracellular antigen binding domain with the second binding, wherein a decrease in the second binding of the tested extracellular antigen binding domain to sialyl Lewis A as compared to the initial binding indicates that the tested extracellular antigen binding domain cross-competes with the reference antibody for binding to sialyl Lewis A, e.g., recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope. In certain embodiments, the tested extracellular antigen binding domain is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, sialyl Lewis A is expressed in cells, e.g., in a flow cytometry assay. In certain embodiments, sialyl Lewis A is immobilized on a surface comprising a Biacore ship (e.g., in a Biacore assay) or other medium suitable for surface plasmon resonance analysis. The binding of the tested extracellular antigen binding domain in the presence of a completely unrelated antibody (not binding to sialyl Lewis A) can serve as a control high value. A control low value can be obtained by incubating the labeled tested extracellular antigen binding domain with the unlabeled tested extracellular antigen binding domain, wherein competition and decreased binding of the labeled tested extracellular antigen binding domain occurs. In certain embodiments, a decrease in the binding of the tested extracellular antigen binding domain to sialyl Lewis A in the presence of the reference antibody 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% is considered an extracellular antigen binding domain that cross-competes with the reference antibody for binding to sialyl Lewis A. In certain embodiments, the assay is performed at room temperature.

[0151] As is well known in the art, the CDR3 domain can independently determine the binding specificity of an antibody or its antigen-binding portion for a cognate antigen, independent of the CDR1 and / or CDR2 domains. Based on a common CDR3 sequence, it is expected that multiple antibodies with the same binding specificity can be generated. See, for example, Klimka et al., British J. of Cancer 83(2):252-260 (2000) (describing the generation of a humanized anti-CD30 antibody using only the CDR3 of the heavy chain variable domain of the murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol. 296:833-849 (2000) (describing a recombinant epithelial glycoprotein 2 (EGP-2) antibody using only the heavy chain CDR3 sequence of the parental murine MOC-31 anti-EGP-2 antibody); Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998) (describing the use of the heavy and light chain variable CDR3 domains of the murine anti-integrin α v β 3 of the antibody LM609 to generate a panel of humanized anti-integrin α v β 3 antibodies, where each member antibody includes a different sequence outside the CDR3 domain and is capable of binding the same epitope as the parental murine antibody with equal or higher affinity); Barbas et al., J. Am. Chem. Soc. 116:2161-2116 (1994) (disclosing that the CDR3 domain makes the most significant contribution to antigen binding); Barbas et al., Proc. Natl. Acad. Sci. USA 92:2529-2533 (1995) (describing the transplantation of the heavy chain CDR3 sequences of three Fabs (SI-1, SI-40, and SI-32) against human placental DNA onto the heavy chain of an anti-tetanus toxoid Fab to replace the existing heavy chain CDR3 and demonstrating that the CDR3 domain alone confers binding specificity); and Ditzel et al., J. Immunol. 157:739-749 (1996) (describing a transplantation study where transferring only the heavy chain CDR3 of the parental multispecific Fab LNA3 onto the heavy chain of the monospecific IgG tetanus toxoid-binding Fab p313 antibody was sufficient to retain the binding specificity of the parental Fab). Each of these references is incorporated herein by reference in its entirety.

[0152] In certain embodiments, the extracellular antigen-binding domain of the CARs described herein includes a heavy-chain variable region CDR3 that includes the amino acid sequence shown in SEQ ID NO: 3, conservative modifications of SEQ ID NO: 3, and / or a light-chain variable region CDR3 that includes the amino acid sequence shown in SEQ ID NO: 6 or conservative modifications thereof. In some such embodiments, the extracellular antigen-binding domain may further (i) include a heavy-chain variable region CDR2 that includes the amino acid sequence shown in SEQ ID NO: 2 or conservative modifications thereof, and a light-chain variable region CDR2 that includes the amino acid sequence shown in SEQ ID NO: 5 or conservative modifications thereof; and / or (ii) include a heavy-chain variable region CDR1 that includes the amino acid sequence shown in SEQ ID NO: 1 or conservative modifications thereof, and a light-chain variable region CDR1 that includes the amino acid sequence shown in SEQ ID NO: 4 or conservative modifications thereof.

[0153] In certain embodiments, the extracellular antigen-binding domain includes a V H CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a V H CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, a V H CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, a V L CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, a V L CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a V L CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0154] In addition, in certain embodiments, the extracellular antigen-binding domain includes: a heavy-chain variable region CDR3 that includes the amino acid sequence shown in SEQ ID NO: 3 or conservative modifications thereof; and a light-chain variable region CDR3 that includes the amino acid sequence shown in SEQ ID NO: 6 or conservative modifications thereof.

[0155] In certain embodiments, the extracellular antigen-binding domain includes: a heavy-chain variable region CDR3 that includes the amino acid sequence shown in SEQ ID NO: 3 or conservative modifications thereof; and a light-chain variable region CDR3 that includes the amino acid sequence shown in SEQ ID NO: 6 or conservative modifications thereof.

[0156] The extracellular antigen-binding domain may further include: a heavy-chain variable region CDR2 that includes the amino acid sequence shown in SEQ ID NO: 2 or conservative modifications thereof; and a light-chain variable region CDR2 that includes the amino acid sequence shown in SEQ ID NO: 5 or conservative modifications thereof.

[0157] In certain embodiments, the extracellular antigen-binding domain comprises: a heavy-chain variable region CDR2 that comprises the amino acid sequence shown in SEQ ID NO: 2 or a conservative modification thereof; and a light-chain variable region CDR2 that comprises the amino acid sequence shown in SEQ ID NO: 5 or a conservative modification thereof.

[0158] The extracellular antigen-binding domain may further comprise: a heavy-chain variable region CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 1 or a conservative modification thereof; and a light-chain variable region CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof.

[0159] In certain embodiments, the extracellular antigen-binding domain comprises: a heavy-chain variable region CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 1 or a conservative modification thereof; and a light-chain variable region CDR1 that comprises the amino acid sequence shown in SEQ ID NO: 4 or a conservative modification thereof.

[0160] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CARs of the present disclosure may comprise a linker that joins the heavy-chain variable region and the light-chain variable region of the extracellular antigen-binding domain. As used herein, the term "linker" refers to a functional group (e.g., chemical or polypeptide) that covalently joins two or more polypeptides or nucleic acids to link them to each other. As used herein, a "peptide linker" refers to one or more amino acids used to join two proteins together (e.g., to link V H and V L domains). In certain embodiments, the linker comprises the amino acids having the sequence shown in SEQ ID NO: 11. In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 is shown in SEQ ID NO: 12.

[0161] Additionally, the extracellular antigen-binding domain may comprise a leader peptide or signal peptide that directs the nascent protein into the endoplasmic reticulum. The signal peptide or leader peptide may be crucial if the CAR is to be glycosylated and anchored in the cell membrane. A signal sequence or leader sequence may be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids in length) present at the N-terminus of a newly synthesized protein that directs them into the secretory pathway. In certain embodiments, the signal peptide is covalently conjugated to the 5' end of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a CD8 polypeptide that comprises the amino acid sequence shown in SEQ ID NO: 13 as provided below.

[0162]

[0163] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 14 provided as follows:

[0164]

[0165] Transmembrane domain of CAR

[0166] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic α-helix spanning at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors aggregate and the signal is transmitted into the cell. According to the subject matter of the present disclosure, the transmembrane domain of the CAR can comprise a natural or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 polypeptide, an NKGD2 polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof.

[0167] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a CD28 polypeptide. In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a human CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof). The CD28 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous (e.g., identical) to the sequence having NCBI reference number P10747 or NP_006130 (SEQ ID NO: 15), and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide can have an amino acid sequence that is a contiguous portion of SEQ ID NO: 15, which is at least 20, or at least 30, or at least 40, or at least 50 and at most 220 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide has 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, or 200 to 220 of SEQ ID NO: 15. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain containing a CD28 polypeptide and an intracellular domain comprising a co-stimulatory signaling region containing a CD28 polypeptide. In certain embodiments, the CD28 polypeptide included in the transmembrane domain and the intracellular domain comprises or has the amino acids 114 to 220 of SEQ ID NO: 15.

[0168] SEQ ID NO: 15 is provided as follows:

[0169]

[0170] According to the subject matter of the present disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide encoding a CD28 polypeptide. An exemplary nucleotide sequence encoding amino acids 114 to 220 of SEQ ID NO: 15 is shown in SEQ ID NO: 16 provided below.

[0171]

[0172] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). The CD28 polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to SEQ ID NO: 17 or a fragment thereof, and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 17, 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. Additionally or alternatively, in various non-limiting embodiments, the CD8 polypeptide comprises or has amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 235 of SEQ ID NO: 17.

[0173]

[0174] According to the subject matter of the present disclosure, a "CD8 nucleic acid molecule" refers to a polynucleotide encoding a CD8 polypeptide.

[0175] In certain embodiments, the transmembrane domain of the CARs of the present disclosure includes the native or modified transmembrane domain of the CD166 polypeptide. The CD166 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having NCBI reference number NP_001618.2 (SEQ ID NO: 18), and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD166 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 18, which is at least 20, or at least 30, or at least 40, or at least 50 and at most 583 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD166 polypeptide comprises or has the amino acid sequence of amino acids 1 to 583, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 450 to 400, 400 to 450, 450 to 500, 528 to 553, 500 to 550, or 500 to 583 of SEQ ID NO: 18. In certain embodiments, the CD166 polypeptide included in the transmembrane domain of the CARs of the present disclosure comprises or has the amino acid sequence of amino acids 528 to 553 of SEQ ID NO: 18.

[0176] SEQ ID NO: 18 is provided below

[0177]

[0178] According to the subject matter of the present disclosure, a "CD166 nucleic acid molecule" refers to a polynucleotide encoding a CD166 polypeptide. An exemplary nucleotide sequence encoding amino acids 528 to 553 of SEQ ID NO: 18 is shown in SEQ ID NO: 19 provided below.

[0179]

[0180] Hinge / spacer region

[0181] In certain non-limiting embodiments, the CAR can further include a hinge / spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The hinge / spacer region can be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition while retaining the activation activity of the CAR. In certain non-limiting embodiments, the hinge / spacer region can be the hinge region of IgG1, the CH 2 CH 3a portion of CD3 and CD28 polypeptide (e.g., SEQ ID NO: 15), a portion of CD8 polypeptide (e.g., SEQ ID NO: 17), a portion of CD166 polypeptide (e.g., SEQ ID NO: 18), a variant having at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology with any of the foregoing, or a synthetic spacer sequence. In certain non-limiting embodiments, the length of the hinge / spacer region can be between about 1-50 (e.g., 5-25, 10-30 or 30-50) amino acids.

[0182] In certain embodiments, the hinge / spacer region of the CAR of the present disclosure includes the native or modified (e.g., conservatively modified) hinge region of the CD166 polypeptide described herein. In certain embodiments, the CD166 polypeptide included in the hinge / spacer region of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 489 to 527 of SEQ ID NO: 18. An exemplary nucleotide sequence encoding amino acids 489 to 527 of SEQ ID NO: 18 is shown as SEQ ID NO: 20 provided below.

[0183]

[0184] Intracellular domain of CAR

[0185] In certain non-limiting embodiments, the intracellular signaling domain of the CAR described herein includes the CD3ζ polypeptide that can activate or stimulate cells (e.g., cells of the lymphoid lineage, such as T cells). Wild-type (“native”) CD3ζ includes three immunoreceptor tyrosine-based activation motifs (“ITAMs”) (e.g., ITAM1, ITAM2, and ITAM3), three basic-rich stretch (BRS) regions (BRS1, BRS2, and BRS3), and after antigen binding, transmits an activation signal to cells (e.g., cells of the lymphoid lineage, such as T cells). The intracellular signaling domain of the native CD3ζ chain is the main sender of signals from the endogenous TCR. As used in the embodiments herein, CD3ζ is not the native CD3ζ, but a modified CD3ζ. In certain embodiments, the intracellular signaling domain of the CAR of the present disclosure includes the CD3ζ polypeptide disclosed in International Patent Application No. PCT / US2018 / 068134, filed on December 31, 2018 (corresponding to International Publication No. WO2019 / 133969), and for the purposes described herein, the content of the foregoing patent application is incorporated herein by reference in its entirety.

[0186] In certain embodiments, the modified CD3ζ polypeptide comprises or has an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the sequence having NCBI reference number NP_932170 (SEQ ID No: 21). In certain non-limiting embodiments, the modified CD3ζ polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 21 and that is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 110, or at least 113, and at most 163 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the modified CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1 to 50, 50 to 100, 100 to 150, 50 to 164, 55 to 164, or 150 to 164 of SEQ ID NO: 21. In certain embodiments, the modified CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 21.

[0187] SEQ ID NO: 21 is provided below:

[0188]

[0189] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified human CD3ζ polypeptide. The modified human CD3ζ polypeptide may comprise or have an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or about 100% homologous (e.g., identical) to SEQ ID NO: 22, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 22 is provided below:

[0190]

[0191] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22 is shown as SEQ ID NO: 23 provided below.

[0192]

[0193] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified human CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises or has an amino acid sequence or a fragment thereof 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% homologous (e.g., identical) to SEQ ID NO: 24, and / or optionally comprises up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 24 is provided below:

[0194]

[0195] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 is shown as SEQ ID NO: 25 provided below.

[0196]

[0197] Immunoreceptor tyrosine - based activation motif (ITAM)

[0198] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises one, two, or three ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM1, which comprises the amino acid sequence shown in SEQ ID NO: 26.

[0199]

[0200] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26 is shown as SEQ ID NO: 27 provided below.

[0201]

[0202] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant that comprises one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide has an ITAM1 variant that comprises two loss-of-function mutations. In certain embodiments, the loss-of-function mutations comprise mutations of tyrosine residues in ITAM1. In certain embodiments, the ITAM1 variant consisting of two loss-of-function mutations comprises the amino acid sequence shown in SEQ ID NO: 28 provided below.

[0203]

[0204] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28 is shown in SEQ ID NO: 29 provided below.

[0205]

[0206] In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM2, which comprises the amino acid sequence shown in SEQ ID NO: 30 provided below.

[0207]

[0208] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30 is shown in SEQ ID NO: 31 provided below.

[0209]

[0210] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant that comprises one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide has an ITAM2 variant that comprises two loss-of-function mutations. In certain embodiments, the loss-of-function mutations comprise mutations of tyrosine residues in ITAM2. In certain embodiments, the ITAM2 variant consisting of two loss-of-function mutations comprises the amino acid sequence shown in SEQ ID NO: 32 provided below.

[0211]

[0212] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32 is shown in SEQ ID NO: 33 provided below.

[0213]

[0214] In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM3, which comprises the amino acid sequence shown in SEQ ID NO: 34 provided below.

[0215]

[0216] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34 is shown in SEQ ID NO: 35 provided below.

[0217]

[0218] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant that includes one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide has an ITAM3 variant that includes two loss-of-function mutations. In certain embodiments, the loss-of-function mutation includes a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant consisting of two loss-of-function mutations includes the amino acid sequence shown in SEQ ID NO: 36 provided below.

[0219]

[0220] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 is shown in SEQ ID NO: 37 provided below.

[0221]

[0222] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises or consists essentially of or consists of an ITAM1 variant that includes one or more loss-of-function mutations, an ITAM2 variant that includes one or more loss-of-function mutations, an ITAM3 variant that includes one or more loss-of-function mutations, or a combination thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises an ITAM2 variant that contains one or more (e.g., two) loss-of-function mutations and an ITAM3 variant that contains one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises native ITAM1, an ITAM2 variant that includes or has two loss-of-function mutations, and an ITAM3 variant that includes or has two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises native ITAM1 having the amino acid sequence shown in SEQ ID NO: 26, an ITAM2 variant having the amino acid sequence shown in SEQ ID NO: 32, and an ITAM3 variant having the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the modified CD3ζ polypeptide comprises or has the amino acid sequence shown in SEQ ID NO: 24.

[0223] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having one or more (e.g., two) loss-of-function mutations and an ITAM3 variant having one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having two loss-of-function mutations, a native ITAM2, and an ITAM3 variant having two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having the amino acid sequence shown in SEQ ID NO: 28, a native ITAM2 having the amino acid sequence shown in SEQ ID NO: 30, and an ITAM3 variant having the amino acid sequence shown in SEQ ID NO: 36.

[0224] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having one or more (e.g., two) loss-of-function mutations and an ITAM2 variant having one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having two loss-of-function mutations, an ITAM2 variant having two loss-of-function mutations, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having the amino acid sequence shown in SEQ ID NO: 28, an ITAM2 variant having the amino acid sequence shown in SEQ ID NO: 32, and a native ITAM3 having the amino acid sequence shown in SEQ ID NO: 34.

[0225] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having two loss-of-function mutations, a native ITAM2, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes an ITAM1 variant having the amino acid sequence shown in SEQ ID NO: 28, a native ITAM2 having the amino acid sequence shown in SEQ ID NO: 30, and a native ITAM3 having the amino acid sequence shown in SEQ ID NO: 34.

[0226] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native ITAM1, native ITAM2, and an ITAM3 variant that includes one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native ITAM1, native ITAM2, and an ITAM1 variant that includes two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native ITAM1 having the amino acid sequence shown in SEQ ID NO: 26, native ITAM2 having the amino acid sequence shown in SEQ ID NO: 30, and an ITAM3 variant having the amino acid sequence shown in SEQ ID NO: 36.

[0227] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native ITAM1, an ITAM2 variant that includes one or more (e.g., two) loss-of-function mutations, and native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native ITAM1, an ITAM2 variant that includes two loss-of-function mutations, and native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native ITAM1 having the amino acid sequence shown in SEQ ID NO: 26, an ITAM2 variant having the amino acid sequence shown in SEQ ID NO: 32, and native ITAM3 having the amino acid sequence shown in SEQ ID NO: 34.

[0228] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes a deletion of one or two ITAMs. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM1 and ITAM2, e.g., the modified CD3ζ polypeptide includes native ITAM3 or an ITAM3 variant and does not include ITAM1 or ITAM2. In certain embodiments, the modified CD3ζ polypeptide includes native ITAM3 having the amino acid sequence shown in SEQ ID NO: 34 and does not include ITAM1 (native or modified) or ITAM2 (native or modified).

[0229] In certain embodiments, the modified CD3ζ polypeptide comprises deletions of ITAM2 and ITAM3. For example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant and does not comprise ITAM2 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM1 having the amino acid sequence shown in SEQ ID NO: 26 and does not comprise ITAM2 (native or modified) or ITAM3 (native or modified).

[0230] In certain embodiments, the modified CD3ζ polypeptide comprises deletions of ITAM1 and ITAM3. For example, the modified CD3ζ polypeptide comprises native ITAM2 or an ITAM2 variant and does not comprise ITAM1 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM2 having the amino acid sequence shown in SEQ ID NO: 30 and does not comprise ITAM1 (native or modified) or ITAM3 (native or modified).

[0231] In certain embodiments, the modified CD3ζ polypeptide comprises a deletion of ITAM1. For example, the modified CD3ζ polypeptide comprises native ITAM2 or an ITAM2 variant and native ITAM3 or an ITAM3 variant and does not comprise ITAM1 (native or modified). In certain embodiments, the modified CD3ζ polypeptide comprises a deletion of ITAM2. For example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant and native ITAM3 or an ITAM3 variant and does not comprise ITAM2 (native or modified). In certain embodiments, the modified CD3ζ polypeptide comprises a deletion of ITAM3. For example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant and native ITAM2 or an ITAM2 variant and does not comprise ITAM3 (native or modified).

[0232] Basic - rich stretch (BRS) region

[0233] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises one, two, or three BRS regions (i.e., BRS1, BRS2, and BRS3). The BRS region can be a native BRS or a modified BRS (e.g., a BRS variant). In certain embodiments, the modified CD3ζ polypeptide comprises the native BRS1 region, which comprises the amino acid sequence shown in SEQ ID NO: 38 provided below.

[0234]

[0235] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38 is shown in SEQ ID NO: 39 provided below.

[0236]

[0237] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS1 variant that includes one or more loss-of-function mutations.

[0238] In certain embodiments, the modified CD3δ polypeptide comprises native BRS2, which includes the amino acid sequence shown in SEQ ID NO: 40.

[0239]

[0240] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40 is shown in SEQ ID NO: 41 provided below.

[0241]

[0242] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS2 variant that contains one or more loss-of-function mutations.

[0243] In certain embodiments, the modified CD3ζ polypeptide comprises native BRS3, which includes the amino acid sequence shown in SEQ ID NO: 42.

[0244]

[0245] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is shown in SEQ ID NO: 43 provided below.

[0246]

[0247] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS3 variant that contains one or more loss-of-function mutations.

[0248] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes all three BRS regions, namely the BRS1, BRS2 region, and BRS3 region. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes native BRS1, native BRS2, and native BRS3.

[0249] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that includes one or two but not all three BRS regions. In certain embodiments, the modified CD3ζ polypeptide includes BRS1 region and BRS2 region and does not include BRS3 region. In certain embodiments, the modified CD3ζ polypeptide includes BRS1 region and BRS3 region and does not include BRS2 region. In certain embodiments, the modified CD3ζ polypeptide includes BRS2 region and BRS3 region and does not include BRS1 region.

[0250] In certain embodiments, the modified CD3ζ polypeptide includes BRS1 region and does not include BRS2 region or BRS3 region. In certain embodiments, the modified CD3ζ polypeptide includes BRS2 region and does not include BRS1 region or BRS3 region. In certain embodiments, the modified CD3ζ polypeptide includes BRS3 region and does not include BRS1 region or BRS2 region.

[0251] In certain embodiments, the modified CD3ζ polypeptide does not include BRS regions (native or modified BRS1, BRS2, or BRS3), e.g., all three BRSs are deleted, e.g., the modified CD3ζ polypeptide included in construct D12.

[0252] In certain non-limiting embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks all or part of the immunoreceptor tyrosine-based activation motif (ITAM), wherein the ITAM is ITAM1, ITAM2, and ITAM3. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM2 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks all or part of the basic-rich stretch (BRS) region, wherein the BRS region is BRS1, BRS2, and BRS3. In certain embodiments, the modified CD3ζ polypeptide lacks BRS2 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM2, ITAM3, BRS2, and BRS3. In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks all or part of the basic-rich stretch (BRS) region, wherein the BRS region is BRS1, BRS2, and BRS3. In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises a BRS variant selected from a BRS1 variant, a BRS2 variant, and a BRS3 variant, wherein the BRS variant comprises one or more loss-of-function mutations.

[0253] Co-stimulatory domain

[0254] In certain non-limiting embodiments, the intracellular domain of the CAR further comprises at least one co-stimulatory signaling region. In certain embodiments, the co-stimulatory signaling region comprises at least one co-stimulatory molecule or a portion thereof, which can provide optimal lymphocyte activation. As used herein, a "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand required for an efficient response of lymphocytes to an antigen. The at least one co-stimulatory signaling region can comprise a CD28 polypeptide (e.g., the intracellular domain of CD28 or a portion thereof), a 4-1BB polypeptide (e.g., the intracellular domain of 4-1BB or a portion thereof), an OX40 polypeptide (e.g., the intracellular domain of OX40 or a portion thereof), an ICOS polypeptide (e.g., the intracellular domain of ICOS or a portion thereof), a DAP-10 polypeptide (e.g., the intracellular domain of DAP-10 or a portion thereof), or a combination thereof. A co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on the cell surface and generates a co-stimulatory response when bound to its receptor, i.e., an intracellular response that provides the stimulation provided when an antigen binds to its CAR molecule. Co-stimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1. As an example, the 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB (also referred to as "CD137") to provide an intracellular signal that, together with the CAR signal, induces the effector cell function of CAR + T cells. CARs comprising an intracellular domain that comprises a co-stimulatory signaling region that comprises 4-1BB, ICOS, or DAP-10 are disclosed in U.S. 7,446,190 (incorporated herein by reference in its entirety, e.g., SEQ ID NO: 15 in U.S. 7,446,190 shows the nucleotide sequence encoding 4-1BB, SEQ ID NO: 16 shows the nucleotide sequence encoding ICOS, and SEQ ID NO: 17 shows the nucleotide sequence encoding DAP-10). In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that contains a CD28 polypeptide. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling region that contains two co-stimulatory molecules, CD28 and 4-IBB or CD28 and OX40.

[0255] 4-1BB can act as a tumor necrosis factor (TNF) ligand and has stimulatory activity. The 4-1BB polypeptide can include an amino acid sequence or a fragment thereof that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homology (e.g., identity) to the sequence having NCBI reference number P41273 or NP_001552 (SEQ ID NO: 44), and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions.

[0256] SEQ ID NO: 44 is provided as follows:

[0257]

[0258] According to the subject matter of the present disclosure, a "4-1BB nucleic acid molecule" refers to a polynucleotide encoding a 4-1BB polypeptide.

[0259] The OX40 polypeptide can have an amino acid sequence or a fragment thereof that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homology (e.g., identity) to the sequence having NCBI reference number P43489 or NP_003318 (SEQ ID NO: 45), and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions.

[0260] SEQ ID NO: 45 is provided as follows:

[0261]

[0262] According to the subject matter of the present disclosure, an "OX40 nucleic acid molecule" refers to a polynucleotide encoding an OX40 polypeptide.

[0263] The ICOS polypeptide can have an amino acid sequence or a fragment thereof that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homology (e.g., identity) to the sequence having NCBI reference number NP_036224 (SEQ ID NO: 46), and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions.

[0264] SEQ ID NO: 46 is provided as follows:

[0265]

[0266] According to the subject matter of the present disclosure, an "ICOS nucleic acid molecule" refers to a polynucleotide encoding an ICOS polypeptide.

[0267] In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the intracellular signaling domain of the CAR includes the intracellular domain of human CD28 or a portion thereof. The CD28 polypeptide may include or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous (e.g., identical) to the amino acid sequence shown in SEQ ID NO: 15, and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide includes or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 15 and is at least 20, or at least 30, or at least 40, or at least 50 and at most 220 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide includes or has 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, or 200 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling region that includes a CD28 polypeptide that includes or has the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 15.

[0268] In certain embodiments, the intracellular signaling domain of the CAR includes the intracellular domain of murine CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide includes or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the sequence having NCBI reference number NP_031668.3 (SEQ ID NO: 47), and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide includes or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 47 and is at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most 218 amino acids in length. Additionally or alternatively, in various non-limiting embodiments, the CD28 polypeptide includes or has the amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 178 to 218, or 200 to 220 of SEQ ID NO: 47. In certain embodiments, the co-stimulatory signaling region of the CAR of the present disclosure includes a CD28 polypeptide that includes or has the amino acid sequence of amino acids 178 to 218 of SEQ ID NO: 47.

[0269] SEQ ID NO: 47 is provided as follows:

[0270]

[0271] According to the subject matter of the present disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide encoding a CD28 polypeptide. An exemplary nucleotide sequence encoding amino acids 178 to 218 of SEQ ID NO: 47 is shown as SEQ ID NO: 48 provided below.

[0272]

[0273] In certain embodiments, the intracellular signaling domain of the CAR comprises the intracellular domain of murine CD28 or a portion thereof. The intracellular domain of murine CD28 may comprise or have an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous (e.g., identical) to SEQ ID NO: 49, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 49 is provided as follows:

[0274]

[0275] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 49 is shown as SEQ ID NO: 50 provided below.

[0276]

[0277] In certain embodiments, the intracellular signaling domain of the CAR comprises the intracellular domain of human CD28 or a portion thereof. The intracellular domain of human CD28 may comprise or have an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous (e.g., identical) to SEQ ID NO: 51, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 51 is provided as follows:

[0278]

[0279] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 51 is shown as SEQ ID NO: 52 provided below.

[0280]

[0281] In certain embodiments, mutation sites and / or junctions between domains / motifs / regions of CARs derived from different proteins are deimmunized. The NetMHC 4.0 Server can be used to predict the immunogenicity of junctions between different CAR parts. For each peptide containing at least one amino acid from the next part, the binding affinity to all alleles of HLA A, B, and C can be predicted. An immunogenicity score can be assigned to each peptide. The immunogenicity score can be calculated using the formula: Immunogenicity score = [(50 - binding affinity) * HLA frequency]. n . n is the number of predictions for each peptide.

[0282] In certain embodiments, the CAR comprises an extracellular antigen-binding domain containing a human scFv that specifically binds to human sialyl Lewis A, a transmembrane domain comprising a CD28 polypeptide, a CD8 polypeptide, or a CD166 polypeptide, and an intracellular domain comprising a wild-type or modified CD3ζ polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide or a 4-1BB polypeptide. The CAR further comprises a signal peptide or leader peptide covalently conjugated to the 5' end of the extracellular antigen-binding domain. The signal peptide comprises the amino acids having the sequence shown in SEQ ID NO: 13. In certain embodiments, the human scFv is scFv 5B1, the variable region sequence of which is provided in Table 1.

[0283] In some embodiments, the CARs of the present disclosure further comprise an inducible promoter for expressing a nucleic acid sequence in human cells. The promoter for expressing the CAR gene can be a constitutive promoter, such as the ubiquitin protein C (UbiC) promoter.

[0284] The subject matter of the present disclosure also provides a nucleic acid molecule encoding a CAR targeting sialyl Lewis A described herein or a functional portion thereof. In certain embodiments, the nucleic acid molecule encodes a CAR targeting sialyl Lewis A of the present disclosure, which comprises a human scFv that specifically binds to human sialyl Lewis A, a transmembrane domain comprising a CD28 polypeptide, a CD8 polypeptide, or a CD166 polypeptide, and an intracellular domain comprising a wild-type or modified CD3ζ polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide or a 4-1BB polypeptide.

[0285] In certain embodiments, a nucleic acid molecule encodes a CAR targeting sialyl Lewis A, which comprises: a human scFv comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7, a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8, and a linker having the amino acid sequence shown in SEQ ID NO: 11 located between the heavy chain variable region and the light chain variable region; a transmembrane domain comprising a CD28 polypeptide, a CD8 polypeptide, or a CD166 polypeptide; and an intracellular domain comprising a wild-type or modified CD3ζ polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide or a 4-1BB polypeptide.

[0286] In certain embodiments, a nucleic acid molecule encodes a functional portion of the CAR targeting sialyl Lewis A of the present disclosure. As used herein, the term "functional portion" refers to any portion, component, or fragment of the CAR targeting sialyl Lewis A of the present disclosure that retains the biological activity of the CAR targeting sialyl Lewis A (the parental CAR). For example, a functional portion includes a portion, component, or fragment of the CAR targeting sialyl Lewis A of the present disclosure that retains the ability to recognize target cells and treat diseases similar to, the same as, or even to a greater extent than the parental CAR. In certain embodiments, an isolated nucleic acid molecule encoding a functional portion of the CAR targeting sialyl Lewis A of the present disclosure can encode a protein comprising, for example, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, or more of the parental CAR.

[0287] V. Immune response cells

[0288] The subject matter of the present disclosure provides cells comprising the CAR targeting sialyl Lewis A of the present disclosure, and methods of using such cells to treat malignant growths, such as treating cancers such as pancreatic cancer. For example, in some embodiments, the present disclosure provides T cells comprising a chimeric antigen receptor that recognizes sialyl Lewis A disclosed herein. Such cells are administered to a human subject in need thereof to treat and / or prevent the malignant growth of tumors such as solid tumors, such as pancreatic cancer.

[0289] In some embodiments, the CARs described herein can be delivered to immune response cells by suitable means known to those skilled in the art. For example, in some embodiments, the CARs described herein can be delivered to immune response cells by a vector or other delivery vehicle. In some embodiments, the CARs described herein can be delivered to immune response cells in the form of an RNA (e.g., mRNA) construct. In some embodiments, immune response cells can be transduced with the CARs of the present disclosure using a viral vector (e.g., a retroviral vector) to cause the cells to express the CAR. The subject matter of the present disclosure also provides methods of using such cells to treat tumors or solid tumors such as pancreatic cancer.

[0290] The immune response cells of the subject matter of the present disclosure can be cells of the lymphoid lineage. The lymphoid lineage, which includes B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of host foreign cells, and the like. Non-limiting examples of immune response cells of the lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells can be differentiated). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of the present disclosure can be any type of T cell, 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 two types of effector memory T cells such as T EM cells and T EMRA cells, regulatory T cells (also known as suppressor T cells), 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 the death of infected somatic cells or tumor cells. A patient's own T cells can be genetically modified to target a specific antigen by introducing any of the polypeptides or systems disclosed herein. The T cell can be a CD4 + T cell or a CD8 + T cell. In certain embodiments, the T cell is a CD4 + T cell. In certain embodiments, the T cell is a CD8 + T cell.

[0291] In certain embodiments, T cells expressing the CAR express Foxp3 to achieve and maintain a T regulatory phenotype.

[0292] In certain embodiments, the cell is a natural killer cell. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and play a role in the innate immune response. NK cells do not require prior activation to perform their cytotoxic function against target cells.

[0293] The immune response cells of the present disclosure may express an extracellular antigen-binding domain (such as a human scFV, an optionally cross-linked Fab, or F(ab) that specifically binds sialyl Lewis A (such as human sialyl Lewis A) 2 ) for treating cancer, such as pancreatic cancer. Such immune response cells can be administered to a subject (such as a human subject) in need thereof to treat cancer. In certain embodiments, the immune response cells are T cells. The T cells can be CD4 + T cells or CD8 + T cells. In certain embodiments, the T cells are CD4 + T cells. In certain embodiments, the T cells are CD8 + T cells.

[0294] The immune response cells of the present disclosure may further include at least one recombinant or exogenous co-stimulatory ligand. For example, the immune response cells of the present disclosure may also be transduced with at least one co-stimulatory ligand to enable the immune response cells to co-express or be induced to co-express a CAR targeting sialyl Lewis A and at least one co-stimulatory ligand. The interaction between the CAR targeting sialyl Lewis A and at least one co-stimulatory ligand provides a non-antigen-specific signal that is important for the full activation of immune response cells (such as T cells). Co-stimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation that stimulates the acute phase response. Its main role is to regulate immune cells. Members of the TNF superfamily have many common characteristics. Most members of the TNF superfamily are synthesized as type II transmembrane proteins (extracellular C-terminus), which contain a short cytoplasmic segment and a relatively long extracellular region. Members of the TNF superfamily include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor β (TNFβ) / lymphotoxin-α (LTα), lymphotoxin-β (LTβ), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large class of cell surface and soluble proteins that are involved in the processes of cell recognition, binding, or adhesion. These proteins share structural characteristics with immunoglobulins - they have immunoglobulin domains (folds). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, which are ligands for CD28, and PD-L1 / (B7-H1), which is a ligand for PD-1. In certain embodiments, at least one co-stimulatory ligand is selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the immune response cell includes a recombinant co-stimulatory ligand, which is 4-1BBL. In certain embodiments, the immune response cell includes two recombinant co-stimulatory ligands, which are 4-1BBL and CD80. Immune response cells comprising a CAR and at least one recombinant co-stimulatory ligand are described in U.S. Patent No. 8,389,282 and U.S. Patent Publication No. 2016 / 0045551, which are incorporated herein by reference in their entirety.In certain embodiments, the immune response cells include a CAR targeting sialyl Lewis A of the present disclosure and a recombinant cytokine (e.g., IL-12). In certain embodiments, the immune response cells include a CAR targeting sialyl Lewis A of the present disclosure and a recombinant CD40L polypeptide.

[0295] In addition, the immune response cells of the present disclosure may further include at least one exogenous cytokine. For example, the immune response cells of the present disclosure can also be transduced with at least one cytokine so that the immune response cells secrete at least one cytokine and express a CAR targeting sialyl Lewis A. In certain embodiments, the at least one cytokine is selected from IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21. In certain embodiments, the cytokine is IL-12.

[0296] Sialyl Lewis A-specific or sialyl Lewis A-targeting human lymphocytes can be used in peripheral donor lymphocytes, such as those disclosed in Sadelaín, M. et al., 2003, Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express a CAR), in Morgan, R.A. et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex including α and β heterodimers), in Panelli, M.C., et al., 2000 J Immunol 164:495-504; Panelli, M.C., et al., 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TIL) in tumor biopsies) and in Dupont, J., et al., 2005 Cancer Res 65:5417-5427; Papanicolaou, G.A., et al., 2003 Blood 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells). The immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0297] In certain embodiments, the immune response cells (e.g., T cells) of the present disclosure express from about 1 to about 5, about 1 to about 4, about 2 to about 5, about 2 to about 4, about 3 to about 5, about 3 to about 4, about 4 to about 5, about 1 to about 2, about 2 to about 3, about 3 to about 4, or about 4 to about 5 vector copies per cell of the CAR of the present disclosure that targets sialyl Lewis A.

[0298] In addition, the immune response cells can comprise and express (e.g., be native or modified to express) an antigen recognition receptor that binds to a second antigen different from sialyl Lewis A (e.g., human sialyl Lewis A). In addition to the CARs of the present disclosure, the inclusion of an antigen recognition receptor on the immune response cells can increase the avidity of the CAR or the immune response cells comprising it for target cells, particularly for CARs with a relatively low binding affinity for sialyl Lewis A (e.g., human sialyl Lewis A), such as, for example, K d is about 2×10 -8 M or higher, about 5×10 -8 M or higher, about 8×10 -8 M or higher, about 9×10 -8 M or higher, about 1×10 -7 M or higher, about 2×10 -7 M or higher, or about 5×10 -7 M or higher.

[0299] In certain embodiments, the antigen recognition receptor is a chimeric costimulatory receptor (CCR). As used herein, the term "chimeric costimulatory receptor" or "CCR" refers to a chimeric receptor that binds an antigen and provides a costimulatory signal but does not provide a T cell activation signal. CCRs are described in Krause et al., J. Exp. Med. (1998); 188(4): 619-626 and US20020018783, the contents of which are incorporated herein by reference in their entirety. CCRs mimic costimulatory signals, but unlike CARs, CCRs do not provide a T cell activation signal, e.g., CCRs lack the CD3ζ polypeptide. CCRs provide costimulation, e.g., CD28-like signals, in the absence of natural costimulatory ligands on antigen-presenting cells. Combinatorial antigen recognition, i.e., the use of CCRs in combination with CARs, can enhance T cell reactivity against T cells expressing dual antigens, thereby improving selective tumor targeting. Kloss et al. described a strategy that integrates combinatorial antigen recognition, separate signal transduction, and critically balanced T cell activation and costimulation strength to generate T cells that eliminate target cells expressing an antigen combination while sparing cells expressing each antigen individually (Kloss et al., Nature Biotechnology (2013); 31(1): 71-75, the contents of which are incorporated herein by reference). In this approach, T cell activation requires CAR-mediated recognition of one antigen (e.g., sialyl Lewis A), while costimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, the combinatorial antigen recognition approach reduces the efficiency of T cell activation to a level that is ineffective without rescue by simultaneous CCR recognition of a second antigen. In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds an antigen different from sialyl Lewis A, a transmembrane domain, and a costimulatory signaling region comprising at least one costimulatory molecule, the costimulatory molecule including but not limited to CD28, 4-1BB, OX40, ICOS, PD-1, CTLA-4, LAG-3, 2B4, and BTLA. In certain embodiments, the costimulatory signaling region of the CCR comprises one costimulatory signaling molecule. In certain embodiments, the one costimulatory signaling molecule is CD28. In certain embodiments, the one costimulatory signaling molecule is 4-1BB. In certain embodiments, the costimulatory signaling region of the CCR comprises two costimulatory signaling molecules. In certain embodiments, the two costimulatory signaling molecules are CD28 and 4-1BB. The second antigen is selected such that the expression of both sialyl Lewis A and the second antigen is restricted to the target cells (e.g., cancer tissue or cancer cells). Similar to CARs, the extracellular antigen-binding domain can be an scFv, Fab, F(ab) 2or a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain. In certain embodiments, the CCR binds to a pancreatic cancer-specific antigen.

[0300] In certain embodiments, the antigen recognition receptor is a truncated CAR. A "truncated CAR" differs from a CAR in that it lacks an intracellular signaling domain. For example, a truncated CAR includes an extracellular antigen-binding domain and a transmembrane domain and lacks an intracellular signaling domain. According to the subject matter of the present disclosure, the truncated CAR has a high binding affinity for a second antigen expressed on a target cell (e.g., a pancreatic cancer cell), such as a pancreatic cancer-specific antigen. The truncated CAR acts as an adhesion molecule, enhancing the avidity of the CARs of the present disclosure, particularly for CARs that have a low binding affinity for sialyl Lewis A, thereby improving the efficacy of the CARs of the present disclosure or immune response cells (e.g., T cells) comprising the same. In certain embodiments, the T cells of the present disclosure comprise or are transduced to express a CAR of the present disclosure targeting sialyl Lewis A and a truncated CAR targeting a pancreatic cancer-specific antigen.

[0301] VI. Nucleic Acid Compositions and Vectors

[0302] The subject matter of the present disclosure provides nucleic acid compositions comprising polynucleotides encoding the CARs disclosed herein. Cells comprising such nucleic acid compositions are also provided.

[0303] Genetic modification of immune response cells (e.g., T cells, NK cells) can be accomplished by delivering a recombinant DNA or RNA construct encoding a CAR to the target cells of a substantially homogeneous cell composition. In some embodiments, such recombinant DNA or RNA constructs can be delivered to immune response cells using a vector. In some embodiments, such a vector can be a retroviral vector (e.g., γ-retrovirus), which is used to introduce the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding a CAR targeting sialyl Lewis A can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter.

[0304] Non-viral vectors or RNAs can also be used. Random chromosomal integration or targeted integration (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPR)) or transgenic expression (e.g., using native or chemically modified RNAs) can be used.

[0305] For the initial genetic modification of cells to provide cells expressing a chimeric antigen receptor (CAR) targeting sialyl Lewis A, retroviral vectors are commonly used for transduction, although any other suitable viral vector or non-viral delivery system can be used. For subsequent genetic modification of cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) has also proven effective. A combination of a retroviral vector and a suitable packaging system is also suitable, wherein the capsid protein will have the function of infecting human cells. A variety of cell lines that produce amphotropic viruses are known, including but not limited to PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, e.g., particles pseudotyped with VSVG, RD114 or GALV envelopes and any other particles known in the art.

[0306] Possible transduction methods also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni et al. (1992) Blood 80:1418-1422, or culture with a separate viral supernatant or concentrated vector stocks in the presence or absence of appropriate growth factors and polycations, e.g., by the methods of Xu et al. (1994) Exp. Hemat. 22:223-230; and Hughes et al. (1992) J. Clin. Invest. 89:1817.

[0307] Transducing viral vectors can be used to express co-stimulatory ligands and / or secrete cytokines (such as 4-1BBL and / or IL-12) in immune response cells. Preferably, the selected vector exhibits high infection efficiency 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; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini 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 can be used include, for example, adenovirus, lentivirus, and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus, or herpes virus, such as Epstein-Barr virus (also see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors have been particularly well developed and have been used clinically (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0308] In certain non-limiting embodiments, the vector expressing the CAR targeting sialyl Lewis A of the present disclosure is a retroviral vector, such as an oncoretroviral vector.

[0309] Non-viral methods can also be used to express proteins in cells. For example, nucleic acid molecules can be introduced into cells by administering nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral gene transfer methods include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes may also be potentially useful for delivering DNA into cells. Transplantation of normal genes into affected tissues of a subject can also be accomplished by ex vivo transfer of normal nucleic acids into a culturable cell type (e.g., autologous or heterologous primary cells or their progeny), followed by injection of the cells (or their progeny) into the target tissue or systemic injection. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression can be achieved by RNA electroporation.

[0310] cDNA expression for polynucleotide therapy can be directed from any suitable promoter (such as the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and regulated by any appropriate mammalian regulatory element or intron (such as the elongation factor 1α enhancer / promoter / intron construct). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of the nucleic acid. The enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, if genomic clones are used as therapeutic constructs, regulation can be mediated by homologous regulatory sequences or, if desired, by regulatory sequences derived from heterologous sources, including any of the above promoters or regulatory elements. The resulting cells can be grown under conditions similar to those of unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

[0311] VII. Genomic integration into immune-responsive cells

[0312] In certain embodiments, the sialyl Lewis A-targeted CAR of the present disclosure can be integrated into a selected locus of the genome of an immune-responsive cell. Any targeted genome editing method can be used to integrate the CAR into a selected locus of the immune-responsive cell genome. In certain embodiments, the expression of the sialyl Lewis A-targeted CAR of the present disclosure is driven by an endogenous promoter / enhancer within or near the locus. In certain embodiments, the expression of the sialyl Lewis A-targeted CAR of the present disclosure is driven by an exogenous promoter integrated into the locus. The locus for integrating the sialyl Lewis A-targeted CAR of the present disclosure is selected based on the expression level of the gene within the locus and the timing of gene expression of the gene within the locus. The expression level and timing may vary at different stages of cell differentiation and in the mitogen / cytokine microenvironment, which are factors to be considered in the selection.

[0313] In certain embodiments, a CRISPR system is used to integrate the disclosed sialyl Lewis A-targeting CAR into a selected locus of the genome of an immune response cell. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to direct it to the correct segment of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin loop) to form an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional segment of a DNA repair template (DNA that guides the cell repair process to allow insertion of a specific DNA sequence). CRISPR / Cas9 typically uses plasmid transfection of target cells. The crRNA needs to be designed for each application because this is the sequence that Cas9 uses to recognize and directly bind to the target DNA in the cell. The repair template carrying the CAR expression cassette also needs to be designed for each application because it must overlap with the sequences flanking the incision and encode the inserted sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). The sgRNA can be ligated to the Cas9 gene and made into a plasmid for transfection into cells. Methods of using the CRISPR system are described, for example, in WO 2014093661 A2, WO 2015123339 A1, and WO2015089354 A1, which are incorporated herein by reference in their entirety.

[0314] In certain embodiments, zinc finger nucleases are used to integrate the disclosed CAR targeting sialyl Lewis A into a selected locus of the genome of an immune response cell. Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing zinc finger DNA-binding domains to DNA cleavage domains. The zinc finger domains can be engineered to target specific DNA sequences, which allows the ZFNs to target desired sequences within the genome. The DNA-binding domains of individual ZFNs typically contain multiple, independently acting zinc finger repeats and can each recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger "modules" with known specificities. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template bearing the CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. When the target sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template and then replicates the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome. Methods using the ZFN system are described, for example, in WO2009146179 A1, WO 2008060510 A2, and CN 102174576 A, which are incorporated herein by reference in their entirety.

[0315] In certain embodiments, a TALEN system is used to integrate the sialyl Lewis A-targeted CAR of the present disclosure into a selected locus of the genome of an immune response cell. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific sequences of DNA. The TALEN system works in much the same way as ZFNs. They are created by fusing a transcription activator-like effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) consist of 33-34 amino acid repeat motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling an array of these TALEs, the TALE DNA-binding domain can be engineered to bind to a desired DNA sequence, thus guiding the nuclease to cut at a specific location in the genome. Methods using the TALEN system are described, for example, in WO 2014134412 A1, WO 2013163628 A2, and WO 2014040370 A1, which are incorporated herein by reference in their entirety. Methods for delivering genome editing agents can vary as needed. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered by viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrodynamic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, pseudotyping of viral vectors with envelope proteins, cis and trans elements of replicable vectors, herpes simplex virus, and chemical mediators (e.g., oligonucleotides, lipid complexes, polymerosomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0316] Modifications can be made anywhere within the selected locus or anywhere that can affect the gene expression of the sialyl Lewis A-targeted CAR being integrated. In certain embodiments, the modification is introduced upstream of the transcription start site of the integrated sialyl Lewis A-targeted CAR. In certain embodiments, the modification is introduced between the transcription start site and the protein-coding region of the integrated sialyl Lewis A-targeted CAR. In certain embodiments, the modification is introduced downstream of the protein-coding region of the sialyl Lewis A-targeted CAR of the present disclosure being integrated.

[0317] VIII. Polypeptides and Analogs and Polynucleotides

[0318] The subject matter of the present disclosure also includes those that bind to sialyl Lewis A (e.g., scFv (e.g., human scFv), Fab, or (Fab) 2) extracellular antigen-binding domains that specifically bind to polypeptides or fragments thereof such as CD3ζ, CD8, CD28, and polynucleotides encoding them, which are modified in a manner that enhances their anti-tumor activity when expressed in immune response cells. The subject matter of the present disclosure provides methods for optimizing amino acid sequences or nucleic acid sequences by generating sequence alterations. Such alterations can include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure also includes analogs of any naturally occurring polypeptides of the subject matter of the present disclosure. The analogs may differ from the naturally occurring polypeptides disclosed herein in terms of amino acid sequence differences, post-translational modifications, or both. The analogs of the subject matter of the present disclosure generally may 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 identity or homology to all or part of the naturally occurring amino acid sequence of the subject matter of the present disclosure. The length of the sequence comparison is at least about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Similarly, in an exemplary method for determining the degree of identity, the BLAST program can be used, and a probability score between e -3 and e -100 indicates closely related sequences. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications can occur during polypeptide synthesis or processing or after treatment with isolated modifying enzymes. The analogs may also differ from the naturally occurring polypeptides of the subject matter of the present disclosure in terms of alterations in the primary sequence. These include natural and induced genetic variants (e.g., caused by random mutagenesis by radiation or exposure to ethyl methanesulfonate or by site-directed mutagenesis as described in the following references: Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd Edition), CSH press, 1989 or Ausubel et al., ibid.). Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids, or non-naturally occurring or synthetic amino acids, such as β or γ amino acids.

[0319] In addition to the full-length polypeptides, the subject matter of the present disclosure also provides fragments of any of the polypeptides or peptide domains of the present disclosure. The fragments can be at least about 5, about 10, about 13, or about 15 amino acids. In some embodiments, the fragment is at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, the fragment is at least about 60 to about 80, about 100, about 200, about 300, or more contiguous amino acids. Fragments of the subject matter of the present disclosure can be generated by methods known to those skilled in the art or can be derived from normal protein processing (e.g., removal of amino acids not required for biological activity from nascent polypeptides, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0320] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins of the invention. Such analogs are administered according to the methods of the subject matter of the present disclosure. Such analogs may exceed the physiological activity of the original polypeptide. Methods for the design of analogs are well known in the art, and analogs can be synthesized according to such methods by modifying the chemical structure such that the resulting analog, when expressed in immune response cells, increases the anti-tumor activity of the original polypeptide. These chemical modifications include, but are not limited to, substitution of alternative R groups and alteration of the saturation at specific carbon atoms of the reference polypeptide. Protein analogs may have relative resistance to in vivo degradation, resulting in a more persistent therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the following examples.

[0321] According to the subject matter of the present disclosure, polynucleotides encoding extracellular antigen-binding domains that specifically bind to sialyl Lewis A (e.g., human sialyl Lewis A) (e.g., scFv (e.g., human scFv), Fab, or (Fab) 2 )、CD3ζ、CD8、CD28 can be modified by codon optimization. Codon optimization can alter both naturally occurring gene sequences and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system. Factors involved in different stages of protein expression include codon adaptation, mRNA structure, and various cis elements in transcription and translation. Any suitable codon optimization method or technique known to those skilled in the art can be used to modify the polynucleotides of the subject matter of the present disclosure, including but not limited to OptimumGene TM 、Encor optimization, and Blue Heron.

[0322] IX. Administration

[0323] The CAR targeting sialyl Lewis A of the present disclosure and immune response cells comprising the same can be administered systemically or directly to a subject to treat or prevent tumor formation. In certain embodiments, the CAR targeting sialyl Lewis A and immune response cells comprising the same are directly injected into an organ of interest (e.g., an organ affected by tumor formation). Additionally or alternatively, for example, by administering to the circulatory system (e.g., tumor vasculature), the CAR targeting sialyl Lewis A and immune response cells comprising the same are indirectly provided to an organ of interest. Expansion and differentiation agents can be provided before, during, or after administration of the cells and compositions to increase the production of T cells in vitro or in vivo.

[0324] The CAR targeting sialyl Lewis A of the present disclosure and immune response cells comprising the same can generally be administered intravascularly in any physiologically acceptable carrier, although they can also be introduced into bone or other convenient sites where the cells can find suitable sites for regeneration and differentiation (e.g., thymus). In certain embodiments, at least 1×10 5 cells can be administered, ultimately reaching about 1×10 10 or more. In certain embodiments, at least 1×10 6 cells can be administered. A cell population comprising immune response cells comprising the CAR targeting sialyl Lewis A of the present disclosure can comprise a purified cell population. A person skilled in the art can readily determine the percentage of immune response cells in a cell population using various well-known methods, such as fluorescence-activated cell sorting (FACS). The purity in a cell population comprising immune response cells comprising the anti-sialyl Lewis A specific CAR of the present disclosure ranges from about 50% to about 55%, about 55% to about 60%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dose can be readily adjusted by a person skilled in the art (e.g., a decrease in purity may require an increase in dose). The immune response cells can be introduced by injection, catheter, etc. If desired, factors can also be included, including but not limited to interleukins such as IL-2, IL-3, IL6, IL-11, IL-7, IL-12, IL-15, IL-21, and other interleukins, colony-stimulating factors such as G-, M-, and GM-CSF, interferons such as gamma-interferon.

[0325] In certain embodiments, the compositions of the present disclosure include a pharmaceutical composition and a pharmaceutically acceptable carrier, the pharmaceutical composition comprising immune response cells comprising a chimeric antigen receptor (CAR) targeting sialyl Lewis A of the present disclosure. Administration can be autologous or allogeneic. For example, immune response cells comprising a CAR targeting sialyl Lewis A of the present disclosure and compositions comprising the same can be obtained from one subject and administered to the same subject or a different compatible subject. T cells of the present disclosure obtained from peripheral blood or their progeny (e.g., of in vivo, ex vivo, or in vitro origin) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering the pharmaceutical compositions of the present disclosure (e.g., a pharmaceutical composition comprising immune response cells comprising a CAR targeting sialyl Lewis A of the present disclosure), they can be formulated in a unit dose injectable form (solution, suspension, emulsion).

[0326] In certain embodiments, the compositions of the present disclosure can include one or more antigen-binding proteins, such as an anti-sialyl Lewis A antibody or an antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.

[0327] XI. Formulations

[0328] Immune response cells comprising a CAR targeting sialyl Lewis A of the present disclosure and compositions comprising the same can be conveniently provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue. The liquid or viscous composition can include a carrier, which can be a solvent or a dispersion medium, comprising, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0329] A sterile injectable solution can be prepared by incorporating a composition of the present disclosure, such as a composition comprising immune response cells expressing a chimeric antigen receptor (CAR) targeting sialyl Lewis A of the present disclosure, into a desired amount of a suitable solvent and incorporating various amounts of other ingredients as needed. Such a composition can be mixed with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, etc. The composition can also be lyophilized. The composition may contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (such as methylcellulose), pH buffering agents, gelling agents or thickening agents, preservatives, flavoring agents, pigments, etc., depending on the route of administration and the desired formulation. Standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, can be referred to for preparing suitable formulations without undue experimentation, and the above standard text is hereby incorporated herein by reference.

[0330] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. The action of microorganisms can be ensured to be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. The absorption of injectable drug forms can be prolonged by using agents that delay absorption such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used will have to be compatible with the immune response cells expressing a CAR that generally targets sialyl Lewis A of the present disclosure.

[0331] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the present disclosure can be achieved using sodium chloride or other pharmaceutically acceptable reagents such as glucose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly preferred for buffers containing sodium ions.

[0332] If desired, a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level. Methylcellulose can be used because it is readily and economically available and easy to use. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The concentration of the thickening agent can depend on the reagent selected. It is important to use an amount that can achieve the selected viscosity. Obviously, the choice of suitable carrier and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a timed-release form or a liquid-filled form).

[0333] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the viability or efficacy of the immune response cells as described in the subject matter of the present disclosure. This will not pose any problem for those skilled in the fields of chemical and pharmaceutical principles, or these problems can be easily avoided according to the present disclosure and the documents cited herein by referring to standard texts or through simple experiments (without involving excessive experimentation).

[0334] One consideration regarding the therapeutic use of the immune response cells of the subject matter of the present disclosure is the number of cells required to achieve an optimal effect. The number of cells to be administered will vary for the subject being treated. In certain embodiments, about 10 4 to about 10 10 、about 10 5 to about 10 9 、or about 10 6 to about 10 8 immune response cells of the subject matter of the present disclosure are administered to a subject. More effective cells can be administered in smaller numbers. In some embodiments, at least about 1×10 8 、about 2×10 8 、about 3×10 8 、about 4×10 8 and about 5×10 8 immune response cells of the subject matter of the present disclosure are administered to a human subject. The precise determination of what will be considered an effective dose can be made based on the individual factors of each subject, including their size, age, gender, weight, and the condition of the specific subject. Those skilled in the art can readily determine the dose from the present disclosure and knowledge in the art.

[0335] One of ordinary skill in the art can readily determine the amounts of the cells and optional additives, vehicles, and / or carriers to be administered in the compositions and in the methods of the present disclosure. Generally, any additives (other than the active cells and / or reagents) are present in phosphate buffered saline in amounts from about 0.001% to about 50% (by weight) solution, and the active ingredient is present in the microgram to milligram range, such as from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt%, from about 0.01 wt% to about 10 wt%, or from about 0.05 wt% to about 5 wt%. For any composition to be administered to an animal or human, and for any particular method of administration, it is necessary to determine toxicity, for example by determining: the lethal dose (LD) and LD50 in a suitable animal model such as a rodent, e.g., a mouse; and the dose of the composition, the concentration of the components therein, and the timing of administration of the composition that elicits a suitable response. Such determination does not require undue experimentation, based on the knowledge of one of ordinary skill in the art, the present disclosure, and the literature cited herein. Also, the duration of continuous administration can be determined without undue experimentation.

[0336] XII. Methods of Treatment

[0337] Methods for treating a malignant growth in a subject are provided herein. The method comprises administering to the subject an amount of the cells of the present disclosure comprising one or more CARs described herein effective to achieve the desired effect, whether alleviating an existing condition or preventing recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be administered in one or more doses. The effective amount can be provided by bolus or by continuous infusion.

[0338] For adoptive immunotherapy using antigen-specific T cells, typically a cell dose in the range of about 10 6 to about 10 11 (e.g., about 10 9 or about 10 6 ) is infused. When immune response cells are administered to a subject and subsequently differentiated, immune response cells specific for a particular antigen (e.g., sialyl Lewis A) are induced. "Induction" of T cells can include, for example, inactivation of antigen-specific T cells by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance in, for example, autoimmune diseases. The immune response cells of the present disclosure can be administered by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, the immune response cells and compositions comprising them are administered intravenously to a subject in need thereof.

[0339] The subject matter of the present disclosure provides various methods of using immune response cells (e.g., T cells) comprising a chimeric antigen receptor (CAR) targeting sialyl Lewis A of the present disclosure. For example, the subject matter of the present disclosure provides methods of reducing the tumor burden of a subject. In certain non-limiting embodiments, the method of reducing the tumor burden comprises administering to the subject an effective amount of the immune response cells of the present disclosure. The immune response cells of the present disclosure can reduce the number of tumor cells, reduce the size of the tumor, and / or eradicate the tumor in the subject.

[0340] The subject matter of the present disclosure also provides methods of increasing or prolonging the survival of a subject suffering from neoplasia. In certain non-limiting embodiments, the method of increasing or prolonging the survival of a subject suffering from neoplasia comprises administering to the subject an effective amount of the immune response cells of the present disclosure. The method can reduce or eradicate the tumor burden of the subject.

[0341] The subject matter of the present disclosure also provides methods for treating and / or preventing neoplasia in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the immune response cells of the present disclosure.

[0342] Cancers that can have their growth inhibited using the immune response cells of the subject matter of the present disclosure include cancers that are generally responsive to immunotherapy. Non-limiting examples of neoplasia, cancers, and / or tumors for treatment include pancreatic cancer.

[0343] Additionally, the subject matter of the present disclosure provides methods of increasing the production of immune activating cytokines in response to cancer cells in a subject. In certain embodiments, the method comprises administering to the subject the immune response cells of the present disclosure. The immune activating cytokines can be granulocyte macrophage colony stimulating factor (GM-CSF), IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, interferon regulatory factor 7 (IRF7), and combinations thereof. In certain embodiments, immune response cells comprising the sialyl Lewis A-specific CAR of the subject matter of the present disclosure increase the production of GM-CSF, IFN-γ, and / or TNF-α.

[0344] Human subjects suitable for therapy typically include two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those with a clinically measurable tumor. A clinically measurable tumor is one that can be detected based on the tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or x-ray; a positive biochemical or histopathological marker alone is not sufficient to identify this group). The pharmaceutical compositions of the present disclosure are administered to these subjects to elicit an anti-tumor response, with the aim of alleviating their symptoms. Ideally, the result is a reduction in the tumor mass, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduced risk or rate of progression or a reduction in the pathological consequences of the tumor.

[0345] A second group of suitable subjects is known in the art as the "adjuvant group". These are individuals with a history of tumor formation but who respond to another form of therapy. Previous therapies can include, but are not limited to, surgical resection, radiotherapy, and conventional chemotherapy. As a result, these individuals do not have a clinically measurable tumor. However, they are suspected of being at risk of developing disease in the vicinity of the primary tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is based on characteristics observed before or after the initial treatment. These characteristics are known in the clinical field and are appropriately defined for each different type of tumor formation. Typical characteristics of the high-risk subgroup are that the tumor has invaded adjacent tissues or shows lymph node involvement. Another group has a genetic susceptibility to tumors but no confirmed clinical signs of tumor formation. For example, a woman who tests positive for a gene mutation associated with breast cancer but is still of childbearing age may wish to receive treatment with one or more of the antigen-binding fragments described herein to prophylactically prevent the occurrence of tumor formation until prophylactic surgery is appropriate.

[0346] The subject may have an advanced form of the disease, in which case the treatment goals may include alleviating or reversing disease progression and / or reducing side effects. The subject may have a history of having been treated, in which case the treatment goals typically include reducing or delaying the risk of recurrence.

[0347] Immune response cells (e.g., T cells) expressing a chimeric antigen receptor (CAR) targeting sialyl Lewis A can be further modified to avoid or minimize immune complications (referred to as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or the risk of outcomes similar to GvHD when healthy tissues express the same target antigen as tumor cells. A potential solution to this problem is to engineer a suicide gene into T cells expressing a CAR targeting sialyl Lewis A. 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) polypeptide. In certain embodiments, the suicide gene is the EGFRt polypeptide. T cell elimination can be achieved by administering an anti-EGFR monoclonal antibody (e.g., cetuximab) to the EGFRt polypeptide. The EGFRt can be covalently conjugated to the 3' end of the intracellular domain of a CAR targeting sialyl Lewis A. The suicide gene can be included in a vector comprising a nucleic acid encoding a CAR targeting sialyl Lewis A of the present disclosure. In this way, during malignant T cell transformation (e.g., GVHD), administration of a prodrug designed to activate the suicide gene (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9)) triggers apoptosis of the CAR-expressing T cells activated by the suicide gene. Incorporating the suicide gene into the CAR targeting sialyl Lewis A of the present disclosure increases the safety level and enables elimination of most CAR T cells within a very short time. The immune response cells (e.g., T cells) of the present disclosure incorporating the suicide gene can be pre-emptively eliminated at a given time point after CAR T cell infusion or eradicated at the earliest signs of toxicity.

[0348] XIII. Kits

[0349] The subject matter of the present disclosure provides kits for treating or preventing neoplasia. In certain embodiments, the kit includes a therapeutic or prophylactic composition that, in unit dose form, includes an effective amount of immune response cells that include a CAR targeting sialyl Lewis A of the present disclosure. In specific embodiments, the cells further express at least one co-stimulatory ligand. In some embodiments, the kit includes a sterile container that contains a therapeutic or prophylactic vaccine; such a container can be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing a medicament.

[0350] If desired, immunoresponsive cells can be provided together with instructions for administering the cells to a subject having or at risk of developing neoplasia. The instructions typically include information regarding the use of the composition for treating and / or preventing neoplasia. In other embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage schedule and administration for treating or preventing neoplasia or its symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (if any), or as a label applied to the container, or provided within or with the container as a separate sheet, booklet, card, or folded printed matter.

[0351] Example

[0352] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the capabilities of those of skill in the art. These techniques are explained fully in the following references: for example, "Molecular Cloning: A Laboratory Manual", 2nd ed. (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); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and are thus to be considered in the preparation and implementation of the present invention. Particularly useful techniques for specific embodiments will be discussed in the following sections.

[0353] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art regarding how to make and use the compositions and the assays, screening and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0354] Example 1

[0355] Introduction

[0356] Strategies to improve antigen presentation, induce epitope spreading, or make existing anti-tumor T cell responses persistent hold promise in combating tumor antigen escape. For example, cancer vaccines and "immunogenic" radiation (RT) activate antigen-presenting cells (APCs) to improve the display of tumor neoantigens to endogenous T cells (Spiotto et al., Sci Immunol (2016); 1). However, the same neoantigens still must be expressed and presented in most, if not all, tumor cells to achieve a complete response. In patients with pre-existing tumor-reactive T cells related to tumor mutation burden, immune checkpoint inhibitors can relieve T cell exhaustion and provide a sustained response. However, checkpoint inhibition cannot restore T cell responses against tumor cells that do not present the recognized antigen, just as CARs cannot direct responses against tumor cells without CAR targets.

[0357] Improved tumor recognition that can occur after exposure to ionizing radiation due to antigen loss, which is mediated by increased APC activation, improved T cell infiltration, and enhanced expression of HLA or CAR targets on tumors (Spiotto et al., Sci Immunol (2016); 1; Weiss et al., Cancer Res (2018); 78:1031 - 1043), faces the same challenge of antigen escape. However, it has been found that tumors exposed to low-dose radiation become more sensitive to CAR T cell activity, including tumor cells lacking CAR targets. Understanding this mechanism may be particularly valuable in overcoming solid tumor antigen escape.

[0358] This alternative mechanism is characterized by enhanced sensitivity of tumors to CAR T cell-mediated elimination through radiation conditioning and utilizes it to expand the scope of action of CAR T cells beyond the target antigen. Over the past few decades, the prognosis of pancreatic cancer has remained poor with little improvement, no uniformly expressed therapeutic target antigens have been established, and the incidence is increasing. In a partial antigen-negative orthotopic pancreatic cancer model, a new method is provided to address the challenge of clonal antigen heterogeneity by combining low-dose radiation and CAR therapy.

[0359] Results

[0360] Sialyl Lewis A (Le A )-specific CAR T cells are active against pancreatic tumor cells in vitro

[0361] Identifying solid tumor targets that are expressed on 100% of tumor cells and not expressed on key normal tissues is challenging. The example of pancreatic cancer illustrates this problem, which has many compelling targets, but none of these targets are clearly expressed on all tumor cells (Zhao et al., Cancer Cell (2015); 28:415 - 428). Sialyl Lewis A (Le A ) is a surface antigen expressed in 75 - 90% of pancreatic tumors (Viola - Villegas et al., J Nucl Med (2013); 54:1876 - 1882), is expressed at low levels on normal human tissues (Viola - Villegas et al., J Nucl Med (2013); 54:1876 - 1882), and is an active antibody target in clinical trials (NCT03118349, NCT02672917, NCT02687230). The human monoclonal 5B1 antibody targeting Le A has been shown to be specific for pancreatic cancer in vitro and in vivo (Viola - Villegas et al., J Nucl Med (2013); 54:1876 - 1882), as well as safe and tolerable in pancreatic cancer patients at biologically active doses (O’Reilly et al., Journal of Clinical Oncology (2017); 35:4110 - 4110). Therefore, a CAR targeting advanced pancreatic ductal adenocarcinoma (APDAC) using this Le A -specific scFv was constructed. The Le A -specific LBBz CAR has effective cytotoxicity against multiple pancreatic cancer tumor cell lines expressing Le A and not against Le A -negative PC3 prostate cancer cells ( Figures 6A - 6C ). Capan2 PDAC expresses moderate levels of Le A ( Figures 6A - 6C ), and was selected for further experiments.

[0362] Low - dose radiation renders tumor cells sensitive to CAR T cell killing without inducing target antigen expression

[0363] To test the initial hypothesis that radiotherapy (RT) can induce LeA expression and enhance the ability of CAR T cells to eliminate tumors with heterogeneous target antigen expression, tumor cells were irradiated with 2 Gy RT, and two days later, cytotoxic T lymphocyte (CTL) assays of the remaining viable cells and FACS analysis of surface target antigen expression were performed. A 2 Gy dose was chosen because higher RT doses induce small but significant tumor cell death, while 2 Gy does not result in detectable differences in tumor viability ( Figure 1A ). It was found that at each effector:target ratio, 2 Gy (hereinafter referred to as "low-dose RT") increased the sensitivity of tumor cells to CAR T cell killing ( Figure 1B ), but surprisingly, did not increase the expression of target antigens ( Figure 1C ).

[0364] Low-dose irradiation affects the gene set associated with sensitivity to TRAIL-mediated death

[0365] To gain insight into the potential mechanism by which low-dose RT sensitizes tumor cells to CAR T cell killing, RNAseq analysis of tumor cells was performed before and after low-dose RT. Although RT itself is sub-lethal, gene set analysis showed that a large number of apoptotic pathways were significantly affected by low-dose RT ( Figure 1D ). In particular, the gene set that differentiates tumor cells sensitive to TRAIL-mediated death from those insensitive to TRAIL-mediated death (Hamai et al., Oncogene (2006); 25: 7618-7634) 1 had the lowest false discovery rate (FDR < 0.0000001 each; inducing 429 out of 492 positive pathway members and downregulating 114 out of 128 negative pathway members) ( Figure 1D ).

[0366] CAR T cells produce TRAIL upon encountering the target antigen

[0367] TRAIL is a trimeric protein that induces death through two different receptors and many downstream signaling molecules that influence sensitivity; tumor cells are generally more sensitive to TRAIL-induced apoptosis than normal cells, but this exists within a certain range (Walczak et al., Nat Med (1999); 5:157-163). Gene set analysis indicates that low-dose RT may transcriptionally initiate TRAIL-mediated death of tumor cells, which is relevant only when the death ligand is locally present at sufficient levels. The production of TRAIL by LeA-specific CAR T cells was analyzed and it was found that CAR T cells produce low levels of TRAIL at baseline, but significantly induce TRAIL mRNA and protein upon encountering the target antigen ( Figure 1E ). In contrast, T cells expressing a truncated CAR lacking the signaling domain (Ldel) do not induce TRAIL after tumor recognition, thus establishing a dependence of TRAIL induction on CAR signaling ( Figure 1F ).

[0368] Antigen-negative tumor cells exposed to low-dose RT are susceptible to CAR T cell TRAIL-mediated death

[0369] To test the functional importance of TRAIL produced by activated CAR T cells on antigen-negative tumors exposed to low-dose RT, tumor cells were FACS sorted into antigen-positive (Ag + ) and antigen-negative (Ag - ) populations. Ag - cells were transduced with firefly luciferase (Luc) and maintained stably antigen-negative over time ( Figure 7 ). A mixture of 75% Ag + and 25% Ag - Luc + tumor cells was exposed to low-dose or no RT and incubated with CAR T cells in which TRAIL was disrupted by CRISPR ( Figures 2A - 2B and Figure 7 ). Three days before TRAIL production was induced, TRAIL wt or knockout CAR T cells were in a resting state or stimulated through their target antigen. Ag - cell killing was monitored using Luc activity, and it was found that wtCAR T cells pre-stimulated on RT-exposed tumor cells produced maximal Ag - tumor cell death, which was significantly reduced by the absence of TRAIL in CAR T cells or the absence of sensitizing RT to the tumor ( Figure 2B)。If L(del)CAR T cells that recognize target cells but do not induce TRAIL are constitutively expressed TRAIL, they will kill significantly more RT-sensitized Ag - tumor cells( Figure 2C )。

[0370] TRAIL plays many context-dependent roles, including apoptosis and necroptosis of tumor cells and T cells, or pro-tumor effects, including myeloid-derived suppressor cell recruitment through tumor cell NFkB activation (Hartwig et al., Mol Cell (2017); 65:730 - 742e735), or survival, invasion, and metastasis within tumors through Rac1 and Akt activation (von Karstedt et al., Cancer Cell (2015); 27:561 - 573). To better understand how RT-sensitized tumors might respond to the increased TRAIL stimulation provided by CAR T cells, mediators of various downstream TRAIL signaling pathways were studied. Many pathway mediators are regulated by transcription, cleavage, phosphorylation, ubiquitination, or other events, but gene expression analysis can provide general information about the overall pathway activation status. Notably, gene expression changes in RNAseq data before and after sensitizing RT indicated that most individual members of both pro-tumor and anti-tumor mediators downstream of TRAIL were significantly altered by sensitizing RT( Figure 3A ; red or green represents significant changes, and gray represents non-significant changes). Pro-survival, migration, metastasis, and tumor-supportive inflammation TRAIL pathway members were almost uniformly downregulated, while pro-apoptotic molecules were overwhelmingly induced, suggesting that sensitizing RT might prime tumor cells for TRAIL-mediated apoptosis( Figure 3A and Figure 9 ). Since apoptosis and necroptosis levels can be monitored by phosphatidylserine (PS) expression on the cell membrane, real-time video microscopy of cultures containing fluorescent annexin-V antibody was used to examine whether TRAIL produced by CAR T cells induced detectable changes in membrane PS over time in Ag - cells. RT-sensitized Ag - tumor cells were labeled with CellTrace Violet (CTV), then mixed with unlabeled Ag + tumor cells and TRAIL wt or TRAIL - / - CAR T cells. Automated quantification of Ag - tumor cells undergoing apoptosis showed that TRAIL - / - CAR T cells were unable to induce Ag - tumor apoptosis over time, while TRAILwt CAR T cells stably and significantly affected Ag - apoptosis of tumor cells (p < 0.0001, Figure 3B ).

[0371] After sensitizing RT, resistant Ag-containing pancreatic tumors in vivo could be eliminated by CAR T cells - populations

[0372] Next, a mouse model was established for a challenging but common clinical situation of heterogeneous solid tumors partially lacking the target antigen. PDAC consisting of 25% Ag - cells was established in the mouse pancreas and treated with CAR T cells 9 days later ( Figure 4A ). Continuous elimination of Ag + CAR T cells for in situ PDAC could not completely eliminate any heterogeneous tumors ( Figures 4B - 4E ). Next, it was tested whether sensitizing RT provided any meaningful benefit in vivo for heterogeneous tumors treated with CAR T cells. Mice with established heterogeneous PDAC, treated with sensitizing RT and then with CAR T cells, obtained more CRs and PRs by imaging, autopsy, and pathological examination ( Figure 4B and 4F ). Since the major known mechanism of CAR-independent T cell killing is through the T cell receptor (TCR), and RT can induce HLA expression on target cells, it was tested whether TCR-dependent tumor killing played an important role after sensitizing RT. CAR T cells lacking TCR (TCR - / - ) ( Figure 10 ) retained the ability to eliminate RT-sensitized heterogeneous tumors ( Figure 4G ). In the first two weeks, RT initially led to a moderate increase in intratumoral T cell accumulation ( Figures 4I - 4K ). Despite a large influx of tumors ( Figure 11 ), TRAIL - / - CAR T cells still failed to achieve a complete response continuously in RT-sensitized tumor-bearing mice, as confirmed by waterfall plots of responses at death (caused by GVHD or tumor progression) ( Figure 4B ) and weekly bioluminescence imaging ( Figure 4H ). By FACS assessment, mice with recurrent / progressive tumors still carried CAR T cells in the blood, spleen, and tumor and showed significant T cell penetration into the tumor by IHC, but showed outgrowth of Ag - tumor cells ( Figure 4L and Figures 12A - 12B ).

[0373] To separate the effects of TRAIL and CAR, RT-sensitized mice were treated with L(del)CAR T cells (which bind to tumors but do not induce CAR cytotoxicity or TRAIL upon recognition) and L(del)-TRAIL CAR T cells (which bind to tumors and constitutively express TRAIL but do not exert CAR-mediated cytotoxicity). Despite local T cell accumulation with the first strategy, no response was generated ( Figure 4M ), but targeting tumors with T cells expressing constitutive TRAIL using an external CAR domain moderately increased the response rate ( Figure 4M ).

[0374] Local RT effectively conditioned the tumors for subsequent CAR T cell administration

[0375] To determine whether systemic RT was required for CAR T cell sensitization or whether local RT to the tumors was sufficient, mice with orthotopic PDAC were treated with RT either systemically or to the pancreatic tumors only, followed by CAR T cell administration ( Figure 5A ). Although mice treated with systemic RT tended to have higher T cell tumor infiltration at early time points ( Figure 13 ), both strategies resulted in similar tumor responses ( Figures 5B - 5D ). Thus, although there may be different host effects between systemic and local low-dose RT, both methods effectively sensitize heterogeneous tumors to CAR T cell killing.

[0376] RT and CAR T cell treatment in patients with heterogeneous tumors: A case report

[0377] The experience of combining RT with CAR T cells is limited. Just as in cell culture and mouse studies, tumor cells that transcriptionally prime TRAIL-mediated killing by RT show significantly more death in response to CAR T cells, it is conceivable that a similar sensitization may occur in adjacent antigen-negative normal tissue cells after RT. A patient with refractory diffuse large B cell lymphoma (DLBCL) with a large proportion of CD19 - tumor cells ( Figures 5E - 5F ) in the sampled tumor mass received CD19 CAR therapy (NCT02631044). The patient had a painful disease infiltrating the skin of his lower leg, especially the right lower leg. Palliative RT (4 Gy × 5 fractions) was provided to his right leg, and then the patient received CD19 CAR T cells as planned. In the days and weeks after CAR T cell therapy, the patient did not show signs or symptoms of toxicity in the irradiated area. The patient presented with grade 2 CRS without neurological symptoms. One month after CAR T cells, the patient had an excellent response by PET-CT imagingFigure 5G ) Two months after CAR T cell infusion, the tumor rebounded at previous and new sites with low / negative CD19 expression, except in the diseased areas that had received palliative RT prior to CAR T cells. Currently, at one year post-treatment, the antigenically heterogeneous tumor areas that received palliative RT and subsequently CAR T cells remain in CR( Figure 5G )

[0378] Discussion

[0379] The initial selection of CD19 as a target in B cell malignancies was largely driven by the elevated and relatively homogeneous expression of CD19 in leukemias and lymphomas and its restriction to the B cell lineage in normal tissues (Brentjens et al., Nat Med (2003); 9:279 - 286; Maher et al., Nat Biotechnol (2002); 20:70 - 75). Based on the significant complete remission rates of 70 - 90% in patients in phase I ALL trials (Sadelain, J Clin Invest (2015); 125:3392 - 3400), the prospect of extending CAR therapy to a wide range of cancers is fascinating. Although CAR therapy has only recently begun to address solid tumors (Zhao et al., Cancer Cell (2015); 28:415 - 428; Morello et al., Cancer Discov (2016); 6:133 - 146; Jindal et al., Med Oncol 2018; 35:87), the results have been modest so far and major responses are rarely seen (Louis et al., Blood (2011); 118:6050 - 6056; Brown et al., N Engl J Med (2016); 375:2561 - 2569). Escape and regrowth of antigen-negative tumor cells are well-documented resistance mechanisms to CAR therapy currently (Brown et al., N Engl J Med (2016); 375:2561 - 2569; Gardner et al., Blood (2016); 127:2406 - 2410; Jackson and Brentjiens, Cancer Discov (2015); 5:1238 - 1240), and new approaches are needed to enable CAR T cells to effectively prevent antigen escape.

[0380] Early approaches to overcome antigen escape from CAR T cells have been to target two different antigens (Hegde et al., J Clin Invest (2016); 126:3036 - 3052). Another approach has been to use "armored" CARs to recruit endogenous T cells through the secretion of activating cytokines such as IL-18 ((Avanzi et al., Cell Rep (2018); 23:2130 - 2141) or the expression of co-stimulatory ligands (Zhao et al., Cancer Cell (2015); 28:415 - 428). Checkpoint inhibitor therapy has been added to CAR T cells with the aim of rejuvenating CAR T cells and endogenous tumor-reactive T cells (Suarez et al., Oncotarget (2016); 7:34341 - 34355; Cherkassky et al., J Clin Invest (2016); 126:3130 - 3144). However, all of these approaches rely on tumor cells expressing tumor-specific antigens that can be recognized by CARs or TCRs. None of these approaches provide a mechanism by which tumor cells lacking both CAR targets and immunogenic TCR epitopes can be eliminated by T cells.

[0381] The method reported herein describes a mechanism by which tumor cells can be eliminated in trans by CAR T cells regardless of their immunogenicity. Thus, the method is relevant to preemptive antigen escape and is particularly beneficial in tumors with low mutational burden, where the likelihood of neoantigen presentation and recognition is low.

[0382] The spatial and temporal specificity obtained herein depends on the physiological response of CAR T cells and the radiosensitization of tumor cells and is independent of target expression. The observation of TRAIL induction in CAR T cells after encountering tumors ensures active and maximal production in the tumor microenvironment. By targeted RT, the ability to induce TRAIL receptors on Ag + and Ag - tumor cells provides a window of opportunity to enhance the site-specific CAR T cell efficacy against heterogeneous tumors. The effects of this interaction have multiple implications. Systemic and local RT have both been shown to sensitize tumors to CAR T cell killing. Most importantly, in antigen-heterogeneous pancreatic cancer, Ag - tumor cells that would otherwise escape CAR recognition have been shown to be eliminated by CAR T cells in vivo after low-dose RT. In the case of systemic disease, low-dose whole-body radiation can effectively sensitize tumor cells and enable their elimination at lower CAR T cell doses, potentially reducing the risk of cytokine release syndrome while increasing efficacy.

[0383] Early observations that tumor cells are highly sensitive to TRAIL-induced apoptosis relative to normal cells (Walczak et al., Nat Med (1999); 5:157-163) gave rise to enthusiasm for recombinant TRAIL or agonist TRAIL receptor-based therapies. Unfortunately, such therapies have encountered multiple limitations, including short half-life of the TRAIL protein (Ichikawa et al., Nat Med (2001); 7:954-960), reduced apoptotic capacity of bivalent antibodies (Wajant, Cell Death Differ (2015); 22:1727-1741), limited local tumor penetration upon systemic administration, and downstream resistance to apoptosis through tumor gene expression changes (Ichikawa et al., Nat Med (2001); 7:954-960). CAR T cells have many potential advantages as a source of TRAIL, such as tumor-intrinsic concentrated TRAIL synthesis, continuous production as long as tumors and T cells are present, and provision of a native trimeric protein rather than potentially less apoptotic bivalent antibodies (Wajant, Cell Death Differ (2015); 22:1727-1741). Although TRAIL may exert a pro-apoptotic effect on CAR T cells through death receptor 5 (Tschumi et al., J Immunother Cancer (2018); 6:71), conditioning by irradiation before infusion of CAR T cells did not increase this activity.

[0384] In some cases, several other forms of immunotherapy are commonly combined with RT. Ablative high-dose radiation that is "immunogenic" causes tumor death and, in some cases, increased antigen presentation, subsequent T cell activation, and potentially a "distant" or secondary immune response to non-irradiated tumors (Spiotto et al., Sci Immunol (2016); 1). Since the frequency of the abscopal effect is low in clinical practice, harnessing this phenomenon is predictably still an active area of research. Unlike endogenous T cells, CAR T cells do not rely on antigen presentation, and it is not intuitive whether radiation has an immunogenic, immunosuppressive, or irrelevant effect on CAR T cell therapy unless radiation induces the expression of a specific CAR target molecule (Weiss et al., Cancer Res (2018); 78:1031-1043). A fundamentally different type of "immunogenic radiation" in CAR T cell therapy is described: a sub-lethal low-dose radiation renders tumors locally sensitive to trans CAR T cell killing. Unlike ablative radiation, sensitizing radiation is not limited by the location or size of the disease, and given that much lower doses can be applied to a much wider area in patients with diffuse metastases, there is more freedom from RT-related side effects.

[0385] Heterogeneous tumor patients treated with palliative (non-therapeutic) RT prior to CAR T cell therapy showed results consistent with the mouse data, without signs of excessive toxicity. Although this clinical correlation was consistent with the animal findings, the hypothesis was not tested. In particular, the role of RT alone in achieving a durable complete response in his heterogeneous tumors cannot be ignored. However, the radiation dose administered was unable to ablate his tumors ex vivo, which is consistent with the fact that the dose was approximately half of the standard local treatment dose of >45 Gy for gross disease in this type of aggressive lymphoma (Ng et al., International journal of radiation oncology, biology, physics (2018); 100:652-669). In addition, although no toxicity was observed in the RT field in the leg, other normal tissues such as the GI system may show increased RT sensitivity to TRAIL produced by activated CAR T cells (Finnberg et al., Cancer Res (2016); 76:700-712). Trials are planned to introduce RT in combination with CAR T cells to evaluate the impact on clonal antigen heterogeneity, the safety of RT conditioning, and the systemic effect of local RT on CAR T cell-mediated disease responses.

[0386] RT is currently used to some extent to palliate approximately half of metastatic cancer patients and is commonly used as an alternative or as a supplement to surgery to improve local control in almost all non-metastatic cancer types (Miller et al., CA Cancer J Clin (2016); 66:271-289). Implementing CAR therapy within current RT regimens may further enhance local and systemic tumor control. These findings suggest that the integrated implementation of these two therapies ensures coordination among disease management teams.

[0387] These findings support the concept that multimodal CAR therapy conditioned with RT can improve responses in solid tumors. Most importantly, a mechanistic platform is provided that can further enhance engineered T cells to eliminate clonally heterogeneous solid tumors.

[0388] Materials and methods

[0389] Cell culture

[0390] Tumor cells expressing firefly luciferase-GFP have been previously described (Zhao et al., Cancer Cell (2015); 28:415-428). The 293T cell line, H29, and retroviral packaging cell lines were cultured in DMEM supplemented with 10% FCS (Zhao et al., Cancer Cell (2015); 28:415-428). Capan-2 cells were generously provided by Jason S. Lewis (MSKCC) and grown in RPMI supplemented with 10% FCS. Cells were tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza) prior to injection into animals.

[0391] Buffy coats from healthy volunteer donors were obtained from the New York Blood Center. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, then the cells were stimulated with PHA (Sigma) and cultured as previously described (Zhao et al., Cancer Cell (2015); 28:415-428).

[0392] Radiation

[0393] Radiation dose: Unless otherwise stated, all experiments using PDAC were performed with 2 Gy. For in vitro RT studies, unless otherwise stated, all RT sensitization experiments were conducted as follows: Tumor cells were given RT two days prior to tumor analysis or co-culture with T cells.

[0394] Radiation method: Local RT of the pancreas was performed by identifying pancreatic tumors using intraperitoneal contrast and cone-beam CT imaging on an X-Rad 225Cx machine, which combines high-precision cone-beam CT imaging with 3D image-guided radiation treatment under general anesthesia. Local RT was delivered using anterior-posterior or anterior-posterior and lateral beams. Experiments with lower target accuracy (whole-body RT) were performed using a small animal irradiator with an opening in the AP direction.

[0395] Flow cytometry

[0396] Fluorescent dye-conjugated antibodies against CD3 (UCHT1), CD4 (S3.5), CD8 (3B5), DR5 (DJR2-4, PE-conjugated, BioLegend), CD95 (DX2, PE-Cy7-conjugated, BD Biosciences), LeA (7LE, AF405-conjugated, Novus), CD19 (SJ25C1), 41BBL (5F4), and Granzyme B (FGB12, Invitrogen) were used. CAR was detected using Alexa647-conjugated goat anti-human F(ab)2 (ThermoFisher). Flow cytometry was performed on a BD LSRII, and data were analyzed using FlowJo software Ver. 9.5.2 (TreeStar). Fc receptor binding inhibitor antibody human (eBioscience) was used to block Fc receptors. In some cases, CountBright beads (Invitrogen) were added to the samples to count the number of cells.

[0397] TRAIL measurement

[0398] For RNA and ELISA experiments, CAR T cells were exposed to Capan2 expressing the target antigen for 4 hours, then the T cells were removed and cultured alone, and they were re-plated in fresh medium every day. Cells were removed and the expression of TRAIL mRNA was analyzed at given time points, and the culture medium was collected at the end of each day for TRAIL ELISA (MyBiosource MBS335491). qPCR was performed using the TaqMan system (ThermoFisher) with primers Hs00921974 (TRAIL), Hs00366278 (DR5), and Hs04194366 (RPL13A housekeeping).

[0399] RNA extraction and real-time quantitative PCR

[0400] Total RNA was extracted from cells using the RNeasy kit (QIAGEN) according to the manufacturer's instructions. RNA concentration and quality were evaluated by ultraviolet spectroscopy using a NanoDrop spectrophotometer (Themo Fisher Scientific). Superscript III First Strand Synthesis SuperMix (Invitrogen) was used to prepare cDNA using 100 to 200 ng of total RNA, with a volume ratio of random hexamers to oligo dT of 1:1. The completed cDNA synthesis reaction was treated with 2 U RNase H at 37 °C for 20 minutes. Quantitative PCR was performed using ABsolute Blue qPCR SYBR Green Low ROX Mix. PCR assays were run on a QuantStudio(TM) 7Flex System, and C t values were obtained using QuantStudio Real-Time PCR software. Relative changes in gene expression were analyzed using the 2 ΔΔCt method.

[0401] Vector constructs

[0402] 1928ζ and 19BBζ CARs including the SJ25C1 CD19-specific scFv have been previously described (Maher et al., Nat Biotechnol (2002); 20: 70 - 75). LBBz and L28z were constructed by replacing the CD19-specific scFv with the human 5B1 scFv targeting Le A . As previously described, all constructs were designed to express Gaussia luciferase for T cell imaging (Santos et al., Nat Med (2009); 15: 338 - 344). The L(del) mutant was created by removing the intracellular co-stimulatory and signaling domains from the designated construct while retaining the extracellular and transmembrane portions. Constructs expressing TRAIL were created by adding the TRAIL cDNA sequence after the designated CAR and P2A sequence.

[0403] Retrovirus production and transduction

[0404] Plasmids encoding SFGγ-retroviral (RV) vectors (Riviere et al., Proc Natl Acad Sci USA (1995); 92:6733 - 6737) were prepared as previously described (Maher et al., Nat Biotechnol (2002); 20:70 - 75). VSV-G pseudotyped retroviral supernatants derived from transduced gpg29 fibroblasts (H29) were used to generate stable retrovirus-producing cell lines as previously described (Gallardo et al., Blood (1997); 90:952 - 957). T cells were transduced by centrifugation on Retronectin (Takara)-coated plates. In T cell knockout studies, CAR transduction was performed directly after Cas9 / gRNA electroporation as described (Eyquem et al., Nature (2017); 543, 113 - 117).

[0405] Cytotoxic T lymphocyte assay (CTL)

[0406] Using 100% Ag + CTL of tumor cells: The cytotoxicity of CAR-transduced T cells was determined by a standard luciferase-based assay. For the luciferase-based assay, tumor cells expressing firefly luciferase-GFP were used as target cells. Effector and tumor cells were co-cultured in triplicate in black-walled 96- or 384-well plates at the indicated E / T ratios. Separate target cells were plated at the same cell density to determine baseline luciferase expression (T cell-free control). After 18 hours, the luciferase substrate (Bright-Glo, Promega) was added directly to each well. The emitted light was measured by a luminometer plate reader or a Xenogen IVIS imaging system (Xenogen) with Living Image software (Xenogen) to obtain an imaging data set. Lysis was determined as [1 - (RLU sample) / (RLU max)] × 100. Assays were performed using CAR T cells transduced within the previous week.

[0407] Using 75% Ag + , 25% Ag - CTL of tumor cells: In experiments involving pre-stimulated CAR T cells, all CAR T cells were grown at a constant concentration of 1 million cells / ml in the presence of 20 U / ml of IL-2 for 10 - 12 days with reconstitution every other day. Cells were pre-stimulated before CTL by adding CAR T cells to adherent cells containing the target antigen (Le A +Capan2) 1 day before the experiment and removing the T cells by aspiration on the day of the experiment. Then the CAR T cells were combined with RT-sensitized 75% Ag+ Capan2 PDAC was co - cultured in a 48 - well plate at an E:T ratio of 1:3. At pre - specified time points of 4 - day LBBz CAR T - cell culture and 5 - day L(del)CART - cell culture, in addition to the relative number of remaining Ag + and Ag - tumor cells, the percentage killing relative to an untreated control was determined. In experiments specifically quantifying Ag - cell killing, only Ag - cells expressed luciferase.

[0408] In all cytotoxicity assays using RT, RT was given to tumor cells (Capan2), which were grown in culture for two days and then the live cells were incubated with CAR T - cells.

[0409] Video microscopy

[0410] In addition to CAR T - cells and annexin - V 595 (Fisher A13203) in an 8 - well microscopy slide, Ag - cells labeled with CTV (CellTrace Violet, Fisher C34571) were mixed with unlabeled Ag + cells at a ratio of 75% LeA + . Confocal images were acquired every 7 minutes during an 18 - hour culture using an LSM880 confocal microscope (Carl Zeiss) at optimal imaging parameters. Data were 3D - rendered and visualized using Imaris (Bitplane). The percentage killing of LeA - cells was determined at each time point using a custom macro in ImageJ / FUI (NIH), which automatically quantifies total Ag - cells (blue cells) and dead / stained Ag - cells (red and blue double - positive).

[0411] Gene disruption

[0412] Forty - eight hours after the start of T - cell activation, cells were transfected by electroporation of Cas9 mRNA and gRNA using an AgilePulse MAX system (Harvard Apparatus). 3×10 6 cells were mixed with 5 μg of Cas9 and 5 μg of gRNA in a 0.2 - cm cuvette. After electroporation, the cells were diluted into the medium and incubated at 37 °C, 5% CO 2Incubate below. To obtain TCR-negative T cells, TCR-positive T cells were removed 3 - 5 days after gRNA transfection using magnetic biotin - anti - TCRαβ and anti - biotin microbeads and an LS column (Miltenyi Biotech). To obtain TRAIL-negative cells, TRAIL-positive T cells were removed using magnetic PE - anti - TRAIL (R&D, FAB687P) and anti - PE microbeads in an LS column (Miltenyi Biotech). To obtain DR5-negative cells, FACS sorting was performed using PE - anti - DR5 staining.

[0413] For TCR knockout, as described previously 42 , gRNAs targeting sequences in the first exon of the constant chain (TRAC) of the TCRα gene required for TCRα and β assembly and localization to the cell surface were used. TRAIL was performed using a synthetic modified gRNA kit (Synthego). The guide RNAs were resuspended at 1 μg / μl -1 in cytoporation T Buffer (Harvard Apparatus). Cas9 mRNA was synthesized by TriLink Biotechnologies.

[0414] Pancreatic cancer tumor model

[0415] According to a protocol approved by the MSKCC Institutional Animal Care and Use Committee, 8 - to 12 - week - old NOD / SCID / IL - 2Rγ - null (NSG) male mice (Jackson Laboratory) were used. A specified ratio of LeA + and LeA - FACS - sorted Capan2 PDAC tumor cells were injected into the pancreas of NSG mice after surgically opening the mice and exposing the pancreas according to an IRB - approved mouse protocol. Each mouse was injected with 75,000 tumor cells in 50% Matrigel. Mice were randomly assigned to treatments, and the personnel performing the treatments and tumor assessments were blinded to the treatment groups. Tumors were allowed to establish in the pancreas for 9 days and then the mice were treated with RT followed by T cells. Tumor volumes were measured by bioluminescence imaging (BLI) using retro - orbital D - luciferin injection and subsequent IVIS imaging. The tumor burden of each mouse was expressed over time relative to the baseline tumor BLI of that mouse at the start of treatment.

[0416] T cell imaging

[0417] CAR T cells containing Gaussia luciferase were imaged using coelenterazine (3031-10 Coelenterazine-SOL in vivo, Nanolight) injected post-orbitally.

[0418] Transcriptome analysis

[0419] Cells were lysed in Trizol LS (Invitrogen) and then subjected to integrated genomics operations at MSKCC for RNA extraction. After ribogreen quantification and quality control on a Bioanalyzer, library preparation was performed on 500 ng of total RNA using the Truseq Stranded Total RNA Library Preparation chemistry (Illumina) with 6 PCR cycles. Samples were barcoded and run on a Hiseq 2500 1T in 50bp / 50bp paired-end mode using the TruSeq SBS Kit v3 (Illumina). Each sample produced an average of 51 million paired reads, with an average of 58% of mRNA bases.

[0420] The output FASTQ data files were mapped to the target genome using the rnaStar aligner, which maps reads genome-wide and resolves reads across splice junctions. A two-pass mapping method was used, where reads were mapped twice. The first pass mapping used a list of known annotated junctions from Ensemble. Then, the new junctions found in the first pass were added to the known junctions and a second pass mapping was performed (in the second pass, the RemoveNoncanonical flag was used). After mapping, the output SAM file was post-processed using PICARD tools to: add read groups, AddOrReplaceReadGroups, which also sorts the file and converts it to the compressed BAM format. An expression count matrix was calculated from the mapped reads using HTSeq (www-huber.embl.de) and one of several possible gene model databases. Then, the R / Bioconductor package DESeq (www-huber.embl.de) was used to normalize the full dataset and analyze differential expression between sample groups, thus processing the raw count matrix generated by HTSeq.

[0421] For the GSA, the Bioconductor package PIANO (bioconductor.org) was used. The exact call was: gsa.res<-runGSA(fc, geneSetStat="mean", gsc=gsc, gsSizeLim=c(min.gns, max.gns), nPerm=nPerm), where fc==foldChange, min.gns==5, max.gns==1000, nPerm==le4. For GeneSet, the MSigDb from Broad (software.broadinstitute.org) was used. The following sets were used: "cl.all.v4.0.symbols.gmt", "c2.all.v4.0.symbols.gmt", "c3.all.v4.0.symbols.gmt", "c5-1.all.v4.0.symbols.gmt", "c6-l.all.v4.0.symbols.gmt", "c7.all.v4.0.symbols.gmt".

[0422] Statistics

[0423] All experimental data are presented as mean ± s.e.m. No statistical method was used to predetermine the sample size. Unpaired two-tailed t-tests were used to compare the groups. Statistical analysis was performed on GraphPad Prism 7 software.

[0424] Embodiments of the present disclosure

[0425] From the foregoing description, it will be apparent that variations and modifications can be made to the subject matter of the present disclosure to adapt it to various uses and conditions. Such embodiments are also within the scope of the appended claims.

[0426] The recitation of a list of elements in any definition of a variable herein includes defining the variable as any single element or combination (or sub-combination) of the listed elements. The recitation of an embodiment herein includes the embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0427] All patents and publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference. Sequence Listing <110> Memorial Sloan-Kettering Cancer Center Biotechnology Research and Development Co., Ltd. <120> Chimeric Antigen Receptor Targeting Sialyl Lewis A and Its Use <130> 072734.0955 <140> PCT / US2019 / 057017 <141> 2019-10-18 <150> 62 / 748,198 <151> 2018-10-19 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 1 Gly Phe Thr Phe Glu Ala Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 2 Ile Asn Trp Asn Ser Gly Arg Ile 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 3 Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly Ala Glu Phe Glu Tyr 1 5 10 15 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 4 Ser Ser Asn Ile Gly Ser Asn Phe 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 5 Arg Asn Asn 1 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 6 Ala Ala Trp Asp Asp Ser Leu Gly Gly His Tyr Val 1 5 10 <210> 7 <211> 142 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 7 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe 35 40 45 Glu Ala Tyr Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ser Ile Asn Trp Asn Ser Gly Arg Ile Ala Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Phe 100 105 110 Tyr Tyr Cys Ala Lys Asp Ile Arg Arg Phe Ser Thr Gly Gly Ala Glu 115 120 125 Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 <210> 8 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 8 Met Ala Gly Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 20 25 30 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 Gly Ser Asn Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 100 105 110 Asp Ser Leu Gly Gly His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr 115 120 125 Val Leu 130 <210> 9 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 9 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggcgt acagtgccag 60 gtgcagctgg tggagtctgg gggaggctcg gtgcagcctg gcaggtccct gagactctcc 120 tgtgaagcct ctggattcac ctttgaggcc tatgccatgc actgggtccg gcaacctcca 180 gggaagggcc tggagtgggt ctcaagtatt aattggaata gtggtcgcat agcctatgcg 240 gactctgtga agggccgatt caccatctcc agagacaacg ccaggaattc cctgtatctg 300 caaatgaaca gtctgagact tgaggacacg gccttctatt actgtgcaaa agatatacgg 360 aggtttagta ccgggggggc ggagtttgag tactggggcc agggaaccct ggtcaccgtc 420 tcctca 426 <210> 10 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <400> 10 atggccggct tccctctcct cctcaccctc ctcactcact gtgcagggtc ttgggcccag 60 tctgtgctga ctcagccgcc ctcagcgtct gggacccccg ggcagagggt caccatctct 120 tgttctggaa gcagctccaa catcggaagt aattttgtat actggtacca gcagctccca 180 ggaacggccc ccaaactcct catatatagg aataatcagc ggccctcagg ggtccctgac 240 cgattctctg gctccaggtc tggcacctca gcctccctgg ccatcagtgg actccggtcc 300 gaggatgagg ctgattatta ctgtgcagca tgggatgaca gcctgggagg ccattatgtc 360 ttcggaactg ggaccaaggt caccgtcctt 390 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Explanation of artificial sequence: synthetic peptide <400> 11 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 12 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Explanation of artificial sequence: synthetic oligonucleotide <400> 12 ggcggcggcg gatctggagg tggtggctca ggtggcggag gctcc 45 <210> 13 <211> 23 <212> PRT <213> Unknown <220> <223> Explanation of unknown: CD8 signal sequence <400> 13 Thr Ala Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 1 5 10 15 Leu Leu His Ala Ala Arg Pro 20 <210> 14 <211> 69 <212> DNA <213> Unknown <220> <223> Unknown description: CD8 signal sequence <400> 14 actgccatgg ccctgccagt aacggctctg ctgctgccac ttgctctgct cctccatgca 60 gccaggcct 69 <210> 15 <211> 220 <212> PRT <213> Homo sapiens <400> 15 Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 <210> 16 <211> 321 <212> DNA <213> Homo sapiens <400> 16 attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60 catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120 tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180 tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240 atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300 gacttcgcag cctatcgctc c 321 <210> 17 <211> 235 <212> PRT <213> Unknown <220> <223> Explanation of unknown: CD8 sequence <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr 20 25 30 Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser 35 40 45 Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala 50 55 60 Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala 65 70 75 80 Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp 85 90 95 Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 100 105 110 Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 115 120 125 Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 195 200 205 Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 210 215 220 Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val 225 230 235 <210> 18 <211> 583 <212> PRT <213> Homo sapiens <400> 18 Met Glu Ser Lys Gly Ala Ser Ser Cys Arg Leu Leu Phe Cys Leu Leu 1 5 10 15 Ile Ser Ala Thr Val Phe Arg Pro Gly Leu Gly Trp Tyr Thr Val Asn 20 25 30 Ser Ala Tyr Gly Asp Thr Ile Ile Ile Pro Cys Arg Leu Asp Val Pro 35 40 45 Gln Asn Leu Met Phe Gly Lys Trp Lys Tyr Glu Lys Pro Asp Gly Ser 50 55 60 Pro Val Phe Ile Ala Phe Arg Ser Ser Thr Lys Lys Ser Val Gln Tyr 65 70 75 80 Asp Asp Val Pro Glu Tyr Lys Asp Arg Leu Asn Leu Ser Glu Asn Tyr 85 90 95 Thr Leu Ser Ile Ser Asn Ala Arg Ile Ser Asp Glu Lys Arg Phe Val 100 105 110 Cys Met Leu Val Thr Glu Asp Asn Val Phe Glu Ala Pro Thr Ile Val 115 120 125 Lys Val Phe Lys Gln Pro Ser Lys Pro Glu Ile Val Ser Lys Ala Leu 130 135 140 Phe Leu Glu Thr Glu Gln Leu Lys Lys Leu Gly Asp Cys Ile Ser Glu 145 150 155 160 Asp Ser Tyr Pro Asp Gly Asn Ile Thr Trp Tyr Arg Asn Gly Lys Val 165 170 175 Leu His Pro Leu Glu Gly Ala Val Val Ile Ile Phe Lys Lys Glu Met 180 185 190 Asp Pro Val Thr Gln Leu Tyr Thr Met Thr Ser Thr Leu Glu Tyr Lys 195 200 205 Thr Thr Lys Ala Asp Ile Gln Met Pro Phe Thr Cys Ser Val Thr Tyr 210 215 220 Tyr Gly Pro Ser Gly Gln Lys Thr Ile His Ser Glu Gln Ala Val Phe 225 230 235 240 Asp Ile Tyr Tyr Pro Thr Glu Gln Val Thr Ile Gln Val Leu Pro Pro 245 250 255 Lys Asn Ala Ile Lys Glu Gly Asp Asn Ile Thr Leu Lys Cys Leu Gly 260 265 270 Asn Gly Asn Pro Pro Pro Glu Glu Phe Leu Phe Tyr Leu Pro Gly Gln 275 280 285 Pro Glu Gly Ile Arg Ser Ser Asn Thr Tyr Thr Leu Thr Asp Val Arg 290 295 300 Arg Asn Ala Thr Gly Asp Tyr Lys Cys Ser Leu Ile Asp Lys Lys Ser 305 310 315 320 Methionine Isoleucine Alanine Serine Threonine Alanine Isoleucine Threonine Valine Histidine Tyrosine Leucine Aspartic acid Leucine Serine Leucine 325 330 335 Asparagine Proline Serine Glycine Glutamic acid Valine Threonine Arginine Glutamine Isoleucine Glycine Aspartic acid Alanine Leucine Proline Valine 340 345 350 Serine Cysteine Threonine Isoleucine Serine Alanine Serine Arginine Asparagine Alanine Threonine Valine Valine Tryptophan Methionine Lysine 355 360 365 Aspartic acid Asparagine Isoleucine Arginine Leucine Arginine Serine Serine Proline Serine Phenylalanine Serine Serine Leucine Histidine Tyrosine 370 375 380 Glutamine Aspartic acid Alanine Glycine Asparagine Tyrosine Valine Cysteine Glutamic acid Threonine Alanine Leucine Glutamine Glutamic acid Valine Glutamic acid 385 390 395 400 Glycine Leucine Lysine Lysine Arginine Glutamic acid Serine Leucine Threonine Leucine Isoleucine Valine Glutamic acid Glycine Lysine Proline 405 410 415 Glutamine Isoleucine Lysine Methionine Threonine Lysine Lysine Threonine Aspartic acid Proline Serine Glycine Leucine Serine Lysine Threonine 420 425 430 Isoleucine Isoleucine Cysteine Histidine Valine Glutamic acid Glycine Phenylalanine Proline Lysine Proline Alanine Isoleucine Glutamine Tryptophan Threonine 435 440 445 Isoleucine Threonine Glycine Serine Glycine Serine Valine Isoleucine Asparagine Glutamine Threonine Glutamic acid Glutamic acid Serine Proline Tyrosine 450 455 460 Isoleucine Asparagine Glycine Arginine Tyrosine Tyrosine Serine Lysine Isoleucine Isoleucine Isoleucine Serine Proline Glutamic acid Glutamic acid Asparagine 465 470 475 480 Val Thr Leu Thr Cys Thr Ala Glu Asn Gln Leu Glu Arg Thr Val Asn 485 490 495 Ser Leu Asn Val Ser Ala Ile Ser Ile Pro Glu His Asp Glu Ala Asp 500 505 510 Glu Ile Ser Asp Glu Asn Arg Glu Lys Val Asn Asp Gln Ala Lys Leu 515 520 525 Ile Val Gly Ile Val Val Gly Leu Leu Leu Ala Ala Leu Val Ala Gly 530 535 540 Val Val Tyr Trp Leu Tyr Met Lys Lys Ser Lys Thr Ala Ser Lys His 545 550 555 560 Val Asn Lys Asp Leu Gly Asn Met Glu Glu Asn Lys Lys Leu Glu Glu 565 570 575 Asn Asn His Lys Thr Glu Ala 580 <210> 19 <211> 78 <212> DNA <213> Homo sapiens <400> 19 ctaattgtgg gaatcgttgt tggtctcctc cttgctgccc ttgttgctgg tgtcgtctac 60 tggctgtaca tgaagaag 78 <210> 20 <211> 116 <212> DNA <213> Homo sapiens <400> 20 accaactgga gagaacagta aactccttga atgtctctgc tataagtatt ccagaacacg 60 atgaggcaga cgagataagt gatgaaaaca gagaaaaggt gaatgaccag gcaaaa 116 <210> 21 <211> 164 <212> PRT <213> Homo sapiens <400> 21 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 100 105 110 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 145 150 155 160 Leu Pro Pro Arg <210> 22 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 22 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 23 <211> 336 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <400> 23 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 24 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 24 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 25 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Explanation of artificial sequence: synthetic polynucleotide <400> 25 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180 gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggccttttc caggggctca gtacagccac caaggacacc 300 ttcgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 26 <211> 22 <212> PRT <213> Unknown <220> <223> Description of unknown: ITAM1 sequence <400> 26 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 1 5 10 15 Asp Val Leu Asp Lys Arg 20 <210> 27 <211> 66 <212> DNA <213> Unknown <220> <223> Description of unknown: ITAM1 sequence <400> 27 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 60 aagaga 66 <210> 28 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 28 Gln Asn Gln Leu Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe 1 5 10 15 Asp Val Leu Asp Lys Arg 20 <210> 29 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic oligonucleotide <400> 29 cagaaccagc tctttaacga gctcaatcta ggacgaagag aggagttcga tgttttggac 60 aagaga 66 <210> 30 <211> 23 <212> PRT <213> Unknown <220> <223> Description of unknown: ITAM2 sequence <400> 30 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 1 5 10 15 Tyr Ser Glu Ile Gly Met Lys 20 <210> 31 <211> 69 <212> DNA <213> Unknown <220> <223> Description of unknown: ITAM2 sequence <400> 31 caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 60 gggatgaaa 69 <210> 32 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 32 Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 1 5 10 15 Phe Ser Glu Ile Gly Met Lys 20 <210> 33 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic oligonucleotide <400> 33 caggaaggcc tgttcaatga actgcagaaa gataagatgg cggaggcctt cagtgagatt 60 gggatgaaa 69 <210> 34 <211> 22 <212> PRT <213> Unknown <220> <223> Description of unknown: ITAM3 sequence <400> 34 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 1 5 10 15 Asp Ala Leu His Met Gln 20 <210> 35 <211> 66 <212> DNA <213> Unknown <220> <223> Unknown description: ITAM3 sequence <400> 35 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 60 atgcag 66 <210> 36 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 36 His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe 1 5 10 15 Asp Ala Leu His Met Gln 20 <210> 37 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic oligonucleotide <400> 37 cacgatggcc ttttccaggg gctcagtaca gccaccaagg acaccttcga cgcccttcac 60 atgcag 66 <210> 38 <211> 5 <212> PRT <213> Unknown <220> <223> Unknown description: BRS1 sequence <400> 38 Lys Arg Arg Gly Arg 1 5 <210> 39 <211> 15 <212> DNA <213> Unknown <220> <223> Unknown description: BRS1 sequence <400> 39 aagagacgtg gccgg 15 <210> 40 <211> 5 <212> PRT <213> Unknown <220> <223> Unknown description: BRS2 sequence <400> 40 Lys Pro Arg Arg Lys 1 5 <210> 41 <211> 15 <212> DNA <213> Unknown <220> <223> Unknown description: BRS2 sequence <400> 41 aagccgagaa ggaag 15 <210> 42 <211> 8 <212> PRT <213> Unknown <220> <223> Unknown description: BRS3 sequence <400> 42 Lys Gly Glu Arg Arg Arg Gly Lys 1 5 <210> 43 <211> 24 <212> DNA <213> Unknown <220> <223> Unknown description: BRS3 sequence <400> 43 aaaggcgagc gccggagggg caag 24 <210> 44 <211> 255 <212> PRT <213> Homo sapiens <400> 44 Methionine, Glycine, Asparagine, Serine, Cysteine, Tyrosine, Asparagine, Isoleucine, Valine, Alanine, Threonine, Leucine, Leucine, Leucine, Valine, Leucine 1 5 10 15 Asparagine, Phenylalanine, Glutamic acid, Arginine, Threonine, Arginine, Serine, Leucine, Glutamine, Aspartic acid, Proline, Cysteine, Serine, Asparagine, Cysteine, Proline 20 25 30 Alanine, Glycine, Threonine, Phenylalanine, Cysteine, Aspartic acid, Asparagine, Asparagine, Arginine, Asparagine, Glutamine, Isoleucine, Cysteine, Serine, Proline, Cysteine 35 40 45 Proline, Proline, Asparagine, Serine, Phenylalanine, Serine, Serine, Alanine, Glycine, Glycine, Glutamine, Arginine, Threonine, Cysteine, Aspartic acid, Isoleucine 50 55 60 Cysteine, Arginine, Glutamine, Cysteine, Lysine, Glycine, Valine, Phenylalanine, Arginine, Threonine, Arginine, Lysine, Glutamic acid, Cysteine, Serine, Serine 65 70 75 80 Threonine, Serine, Asparagine, Alanine, Glutamic acid, Cysteine, Aspartic acid, Cysteine, Threonine, Proline, Glycine, Phenylalanine, Histidine, Cysteine, Leucine, Glycine 85 90 95 Alanine, Glycine, Cysteine, Serine, Methionine, Cysteine, Glutamic acid, Glutamine, Aspartic acid, Cysteine, Lysine, Glutamine, Glycine, Glutamine, Glutamic acid, Leucine 100 105 110 Threonine, Lysine, Lysine, Glycine, Cysteine, Lysine, Aspartic acid, Cysteine, Cysteine, Phenylalanine, Glycine, Threonine, Phenylalanine, Asparagine, Aspartic acid, Glutamine 115 120 125 Lysine, Arginine, Glycine, Isoleucine, Cysteine, Arginine, Proline, Tryptophan, Threonine, Asparagine, Cysteine, Serine, Leucine, Aspartic acid, Glycine, Lysine 130 135 140 Serine, Valine, Leucine, Valine, Asparagine, Glycine, Threonine, Lysine, Glutamic acid, Arginine, Aspartic acid, Valine, Valine, Cysteine, Glycine, Proline 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 45 <211> 277 <212> PRT <213> Homo sapiens <400> 45 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys Ile 275 <210> 46 <211> 199 <212> PRT <213> Homo sapiens <400> 46 Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45 Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu 65 70 75 80 Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu 115 120 125 His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140 Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu 145 150 155 160 Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170 175 Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser 180 185 190 Arg Leu Thr Asp Val Thr Leu 195 <210> 47 <211> 218 <212> PRT <213> Mouse <400> 47 Met Thr Leu Arg Leu Leu Phe Leu Ala Leu Asn Phe Phe Ser Val Gln 1 5 10 15 Val Thr Glu Asn Lys Ile Leu Val Lys Gln Ser Pro Leu Leu Val Val 20 25 30 Asp Ser Asn Glu Val Ser Leu Ser Cys Arg Tyr Ser Tyr Asn Leu Leu 35 40 45 Ala Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asn Ser Asp Val 50 55 60 Glu Val Cys Val Gly Asn Gly Asn Phe Thr Tyr Gln Pro Gln Phe Arg 65 70 75 80 Ser Asn Ala Glu Phe Asn Cys Asp Gly Asp Phe Asp Asn Glu Thr Val 85 90 95 Thr Phe Arg Leu Trp Asn Leu His Val Asn His Thr Asp Ile Tyr Phe 100 105 110 Cys Lys Ile Glu Phe Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Arg 115 120 125 Ser Asn Gly Thr Ile Ile His Ile Lys Glu Lys His Leu Cys His Thr 130 135 140 Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val 145 150 155 160 Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp 165 170 175 Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met 180 185 190 Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala 195 200 205 Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro 210 215 <210> 48 <211> 123 <212> DNA <213> Mouse <400> 48 aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60 gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120 ccc 123 <210> 49 <211> 41 <212> PRT <213> Mouse <400> 49 Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro 20 25 30 Ala Arg Asp Phe Ala Ala Tyr Arg Pro 35 40 <210> 50 <211> 123 <212> DNA <213> Mouse <400> 50 aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60 gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120 ccc 123 <210> 51 <211> 41 <212> PRT <213> Homo sapiens <400> 51 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 52 <211> 123 <212> DNA <213> Homo sapiens <400> 52 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds to sialyl Lewis A, and wherein the extracellular antigen-binding domain comprises: a heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 1; a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 2; a heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 3; a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 4; a light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 5; and a light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

2. The CAR according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

7.

3. The CAR according to claim 2, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

7.

4. The CAR according to claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

8.

5. The CAR according to claim 4, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

6. The CAR according to any one of claims 1-5, wherein: a) the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 7; and b) the light chain variable region comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

8.

7. The CAR according to claim 6, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

8. The CAR according to any one of claims 1-5, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv).

9. The CAR according to claim 8, wherein the scFV is included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

10. The CAR according to any one of claims 1-5, wherein the extracellular antigen-binding domain comprises a human scFv.

11. The CAR according to claim 10, wherein the scFV is included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

12. The CAR according to any one of claims 1-5, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

13. The CAR according to any one of claims 1-5, wherein the extracellular antigen-binding domain comprises a signal peptide covalently conjugated to the 5'-end of the extracellular antigen-binding domain.

14. The CAR according to any one of claims 1-5, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide not based on a protein associated with an immune response, or a combination thereof.

15. The CAR according to any one of claims 1-5, wherein the intracellular domain further comprises at least one co-stimulatory signaling region.

16. The CAR according to claim 15, wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

17. The CAR according to claim 16, wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide.

18. The CAR according to any one of claims 1-5, wherein the intracellular signaling domain comprises a wild-type CD3ζ polypeptide or a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks a) all or part of an immunoreceptor tyrosine-based activation motif (ITAM), wherein the ITAM is ITAM1, ITAM2, and ITAM3; and / or lacks all or part of a basic-rich stretch (BRS) region, wherein the BRS region is BRS1, BRS2, and BRS3.

19. The modified CD3ζ polypeptide according to claim 18, wherein: a) lacks ITAM2 or a portion thereof; b) lacks ITAM1 or a portion thereof; c) lacks ITAM3 or a portion thereof; d) comprises a deletion of ITAM2 or a portion thereof; e) comprises a deletion of ITAM1 or a portion thereof; and / or f) comprises a deletion of ITAM3 or a portion thereof.

20. The CAR according to claim 19, wherein the modified CD3ζ polypeptide: a) lacks ITAM2 or a portion thereof, and further lacks i) ITAM3 or a portion thereof, and / or ii) ITAM1 or a portion thereof.

21. The CAR according to claim 19, wherein the modified CD3ζ polypeptide: b) lacks ITAM1 or a portion thereof, and further lacks ITAM3 or a portion thereof.

22. The CAR according to claim 19, wherein the modified CD3ζ polypeptide: d) comprises a deletion of ITAM2 or a portion thereof, and further comprises i) a deletion of ITAM3 or a portion thereof, and / or ii) a deletion of ITAM1 or a portion thereof.

23. The CAR according to claim 19, wherein the modified CD3ζ polypeptide: e) comprises a deletion of ITAM1 or a portion thereof, and further comprises a deletion of ITAM3 or a portion thereof.

24. The CAR according to claim 18, wherein the modified CD3ζ polypeptide: a) lacks BRS2 or a portion thereof; b) lacks BRS1 or a portion thereof; c) lacks BRS3 or a portion thereof; and / or d) lacks BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof; e) includes a deletion of BRS2 or a portion thereof; f) includes a deletion of BRS1 or a portion thereof; g) includes a deletion of BRS3 or a portion thereof; and / or h) includes a deletion of BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof.

25. The CAR according to claim 24, wherein the modified CD3ζ polypeptide: a) lacks BRS2 or a portion thereof and further lacks i) BRS3 or a portion thereof, and / or ii) BRS1 or a portion thereof.

26. The CAR according to claim 24, wherein the modified CD3ζ polypeptide: b) lacks BRS1 or a portion thereof and further lacks BRS3 or a portion thereof.

27. The CAR according to claim 24, wherein the modified CD3ζ polypeptide: e) includes a deletion of BRS2 or a portion thereof and further includes i) a deletion of BRS3 or a portion thereof, and / or ii) a deletion of BRS1 or a portion thereof.

28. The CAR according to claim 24, wherein the modified CD3ζ polypeptide: f) includes a deletion of BRS1 or a portion thereof and further includes a deletion of BRS3 or a portion thereof.

29. The CAR according to claim 18, wherein the modified CD3ζ polypeptide lacks ITAM2, ITAM3, BRS2, and BRS3, or includes a deletion of ITAM2, ITAM3, BRS2, and BRS3.

30. The CAR according to any one of claims 1 - 5, further comprising a hinge / spacer region, wherein the hinge / spacer region is a native or modified hinge / spacer region of a molecule selected from: CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4 - 1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM - 1 polypeptide, CTLA - 4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof, and wherein the transmembrane domain is a native or modified transmembrane domain of a molecule selected from: CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4 - 1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM - 1 polypeptide, CTLA - 4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof.

31. The CAR according to claim 30, wherein the hinge / spacer region is derived from the same molecule from which the transmembrane domain is derived.

32. The CAR according to claim 31, wherein the CAR comprises: a) The hinge / spacer region of the CD28 polypeptide and the transmembrane domain of the CD28 polypeptide; b) The hinge / spacer region of the CD84 polypeptide and the transmembrane domain of the CD84 polypeptide; c) The hinge / spacer region of the CD166 polypeptide and the transmembrane domain of the CD166 polypeptide; d) The hinge / spacer region of the CD8a polypeptide and the transmembrane domain of the CD8a polypeptide; or e) The hinge / spacer region of the CD8b polypeptide and the transmembrane domain of the CD8b polypeptide.

33. The CAR according to claim 14, wherein the CAR comprises the hinge / spacer region of the CD166 polypeptide and the transmembrane domain of the CD166 polypeptide.

34. The CAR according to claim 15, wherein the transmembrane domain and the hinge / spacer region are derived from different molecules.

35. The CAR according to claim 34, wherein the CAR comprises the hinge / spacer region of the CD28 polypeptide and the transmembrane domain of the ICOS polypeptide.

36. The CAR according to any one of claims 1-5, wherein the CAR is recombinantly expressed or expressed from a vector.

37. The CAR according to claim 36, wherein the vector is a retroviral vector.

38. The CAR according to claim 37, wherein the vector is a γ-retroviral vector.

39. An immune-responsive cell comprising the CAR according to any one of the preceding claims.

40. The immune-responsive cell according to claim 39, wherein the immune-responsive cell is modified using a composition comprising the CAR.

41. The immune-responsive cell according to claim 40, wherein the composition is a vector.

42. The immune-responsive cell according to any one of claims 39-41, wherein the CAR is constitutively expressed on the surface of the immune-responsive cell.

43. The immune-responsive cell according to any one of claims 39-41, wherein the immune-responsive cell is selected from T cells, natural killer (NK) cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursors, and pluripotent stem cells from which lymphoid cells can differentiate.

44. The immune-responsive cell according to claim 43, wherein the immune-responsive cell is a T cell.

45. The immune-responsive cell according to claim 44, wherein the T cell is selected from cytotoxic T lymphocytes (CTLs), regulatory T cells, and central memory T cells.

46. A nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) according to any one of claims 1-38.

47. A vector comprising the nucleic acid molecule according to claim 46.

48. The vector according to claim 47, wherein the vector is a retroviral vector.

49. The vector according to claim 48, wherein the vector is a γ-retroviral vector.

50. A host cell comprising the vector according to any one of claims 47-49, or expressing the nucleic acid molecule according to claim 46.

51. The host cell according to claim 50, wherein the host cell is a T cell.

52. A method for generating an immune response cell that binds to sialyl Lewis A, comprising introducing a nucleic acid molecule comprising a nucleic acid sequence encoding a CAR according to any one of claims 1-38 into the immune response cell.

53. A composition comprising an immune response cell according to any one of claims 39-45.

54. The composition according to claim 53, which is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

55. Use of an effective amount of an immune response cell according to any one of claims 39-45 or a composition according to claim 53 or 54 in the preparation of a medicament for treating or preventing malignant growth in a subject; wherein the malignant growth is pancreatic cancer.

56. The use according to claim 55, wherein the use reduces or eradicates the tumor burden in the subject.

57. The use according to claim 55, wherein the subject is a human.

58. The use according to claim 55, wherein the subject has received a low dose of radiation.

59. A kit for treating or preventing malignant growth, comprising an immune response cell according to any one of claims 39-45.

60. The kit according to claim 59, wherein the kit further comprises written instructions for treating a subject suffering from neoplasia with the immune response cell.

61. The kit according to claim 59, wherein the malignant growth is pancreatic cancer.

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