Compositions and methods for treating cancer with anti-CD33 immunotherapy

Abstract

The present invention relates to compositions and methods for treating cancer with anti-CD33 immunotherapy. The present disclosure relates to chimeric antigen receptors (CARs) comprising CD33 antigen binding domains, including nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions. Also disclosed are methods for treating or preventing cancer in a subject and methods for preparing chimeric antigen receptor T cells. Experimental results of seven different CD33 external domains coupled to a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain derived from a human CD8 protein are provided.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880032715.1. The original application is the PCT international application PCT / US2018 / 024183 filed on March 23, 2018, which entered the Chinese national phase on November 18, 2019.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. Section 119(e) to U.S. Provisional Patent Application No. 62 / 620,139, filed on January 22, 2018, and U.S. Provisional Patent Application No. 62 / 476,438, filed on March 24, 2017, the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on March 20, 2018, is named Sequence_Listing.txt and is 124 kb in size.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made under a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the Department of Health and Human Services. The U.S. Government has certain rights in this invention. Technical Field

[0008] The present application relates to the field of cancer, and in particular to a CD33 antigen-binding domain and a chimeric antigen receptor (CAR) comprising such a CD33 antigen-binding domain and methods of using the same. Background Art

[0009] Cancer is one of the most deadly threats to human health. In the U.S. alone, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after cardiovascular disease, causing approximately one-quarter of all deaths. Solid tumors are the cause of most of these deaths. Although significant progress has been made in the medical treatment of some cancers, the overall 5-year survival rate of all cancers has only improved by approximately 10% over the past 20 years. Cancer or malignant tumors metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult.

[0010] CD33 is a 67 kDa transmembrane cell surface glycoprotein receptor. CD33 is a member of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family. Proteins in this family mediate leukocyte adhesion to endothelial cells by binding to sialylated glycans (Kelm S, Schauer R, Crocker PR. Glycoconj J. 1996; 13: 913-926). In addition, CD33 acts as an inhibitory receptor via an immunoreceptor tyrosine-based inhibitory motif (ITIM). CD33 receptor activation leads to phosphorylation of two tyrosines (Y340 and Y358) in the CD33 cytoplasmic tail, which serve as docking sites for SHP phosphatase and participate in inhibitory signal transduction cascades, such as downregulation of calcium mobilization (Paul SP1, Taylor LS, Stansbury EK, McVicar DW Blood. 2000 Jul 15;96(2):483-90).

[0011] CD33 is a myeloid differentiation antigen and is highly expressed on myeloid progenitor cells (Andrews RG, Torok-Storb B, Bernstein ID. Blood. 1983; 62: 124-132), but is expressed only at low levels in differentiated myeloid cells (i.e., macrophages and granulocytes) (Simmons D, Seed BJ Immunol. 1988; 141: 2797-2800). In contrast, CD33 is reported to be expressed in 87.8%-99% of acute myeloid leukemia (AML) (A Ehninger et al., Blood Cancer Journal (2014) 4, e218; Christina Krupka et al., Blood 2014 123: 356-365). AML is a devastating disease with a 5-year survival rate of approximately 26% (available on the World Wide Web at cancer.net / cancer-types / leukemia-acute-myeloid-aml / statistics). The current standard of care for AML consists of remission induction therapy with high-dose chemotherapy or radiation, followed by consolidation, including allogeneic stem cell transplantation and additional courses of chemotherapy as needed (available on the World Wide Web at cancer.org / cancer / acute-myeloid-leukemia / treating / typical-treatment-of-aml.html). The high toxicity associated with this treatment, as well as the risk of complications such as myelosuppression or GVHD, has prompted the search for better treatment options.

[0012] Many new approaches to treating AML are currently under investigation, including antibody-drug conjugates (SGN-CD33A, Vadastuximab Talirine, Stein AS et al. (2015). Blood, 126(23), 324; Phase I-II clinical trial NCT02706899), bispecific T cell engaging antibodies (AMG330, Laszlo GS et al., Blood 2013: 123(4): 554-561, NCT02520427) and CART-33 cells (Wang QS et al., Mol Ther. 2015 Jan; 23(1): 184-91, NCT01864902). However, many new approaches have been shelved due to clinical toxicity. The Seattle Genetics Phase I clinical trials testing the drug SGN-CD33 were recently put on hold due to the risk of hepatotoxicity (available at businesswire.com / news / home / 20161227005087 / en / Seattle-Genetics-Announc es-Clinical-Hold-Phase-1). Gemtuzumab ozogamicin (Mylotarg, Pfizer / Wyeth) was voluntarily withdrawn from the market by the manufacturer in 2010 after potentially fatal veno-occlusive liver disease was observed in post-marketing clinical trials (Jacob M. Rowe and Bob Blood 2013 121:4838-4841). Although the FDA recently reintroduced Mylotarg for CD33+ adult AML and relapsed / refractory pediatric AML, the drug is prescribed with a new, more conservative lower dose and a new regimen (FDA press release, September 2017, available on the World Wide Web at fda.gov). The efficacy of this treatment, the durability of patients' responses to Mylotarg, the extent of tumor antigen escape, and its safety profile under the new regimen remain to be determined. Therefore, there is an urgent need for safe, effective, and durable treatments for AML.

[0013] Chimeric antigen receptors (CARs) are hybrid molecules that contain three basic units: (1) an extracellular antigen binding motif, (2) a linker / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2(4): e23621). The antigen binding motif of a CAR is typically formed by a single chain variable fragment (ScFv): the minimal binding domain of an immunoglobulin (Ig) molecule. Alternative antigen binding motifs have also been engineered, such as receptor ligands (i.e., IL-13 has been engineered to bind to tumor-expressed IL-13 receptors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or γ-δ T cells) are also under development (Brown CE et al., Clin Cancer Res. 2012; 18(8): 2199-209; Lehner M et al., PLoS One. 2012; 7(2): e31210). There is still a lot of work to define the most active T cell populations for transduction using CAR vectors, determine the best culture and expansion techniques, and define the molecular details of the CAR protein structure itself.

[0014] The connection motif of CAR can be a relatively stable domain (such as the constant domain of IgG) or a flexible linker designed to be extended. Structural motifs (such as those derived from IgG constant domains) can be used to extend the ScFv binding domain away from the T cell plasma membrane surface. This can be important for some tumor targets in which the binding domain is particularly close to the tumor cell surface membrane (such as for disialoganglioside GD2; Orentas et al., unpublished observations). So far, the signal transduction motif for CAR generally includes the CD3-ζ chain because the core motif is a key signal for T cell activation. The second-generation CAR reported for the first time is characterized by CD28 signal transduction domain and CD28 transmembrane sequence. This motif is also used for the third-generation CAR (Zhao Y et al., J Immunol.2009; 183 (9): 5563-74) comprising the CD137 (4-1BB) signal transduction motif. With the advent of new technologies, T cells are activated using beads connected to anti-CD3 and anti-CD28 antibodies, and the presence of the typical "signal 2" from CD28 no longer needs to be encoded by the CAR itself. By using bead activation, it was found that the third-generation vector was not superior to the second-generation vector in in vitro assays, and it did not provide significant benefits relative to the second-generation vector in a mouse model of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, DimitrovDS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ.Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood.2013; 121(7): 1165-74; Kochenderfer JN et al., Blood.2012; 119(12): 2709-20). This is demonstrated by the clinical success of CD19-specific CARs that are second-generation CD28 / CD3-ζ (Lee DW et al., American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD137 / CD3-ζ signaling formats (Porter DL et al., N Engl J Med. 2011; 365(8):725-33).In addition to CD137, other tumor necrosis factor receptor superfamily members (such as OX40) can also provide important sustained signals in CAR-transduced T cells (Yvon E et al., Clin Cancer Res. 2009; 15(18): 5852-60). Equally important are the culture conditions for culturing CAR T cell populations.

[0015] The current challenge in the wider and more effective adaptation of CAR therapy for cancer is related to the lack of powerful targets. Establishing a conjugate of cell surface antigens is now easily achievable, but finding cell surface antigens that are specific to tumors while not harming normal tissues remains a daunting challenge. A potential way to give CAR-expressing T cells greater target cell specificity is to use a combined CAR approach. In one system, CD3-ζ and CD28 signaling units are separated in two different CAR constructs expressed in the same cell; in another system, two CARs are expressed in the same T cell, but one has a lower affinity and therefore requires another CAR to first engage (engage) to make the second fully active (Lanitis E et al., Cancer Immunol Res. 2013; 1 (1): 43-53; Kloss CC et al., Nat Biotechnol. 2013; 31 (1): 71-5). The second challenge in producing a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma in which the effector cell population is simultaneously targeted to multiple antigens (HER2, IL-13Ra, EphA2), hoping to avoid the consequences of a target antigen-negative population (Hegde M et al., Mol Ther. 2013;21(11):2087-101).

[0016] T cell-based immunotherapy has become a new frontier in synthetic biology; a variety of promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where they can not only evade negative regulatory signals but also mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of inducible caspase 9 constructs with AP1903 suggests a way in which a powerful switch that can control T cell populations can be pharmacologically turned on (Di Stasi A et al., N Engl J Med. 2011; 365(18): 1673-83). Generation of effector T cell populations immune to the negative regulatory effects of transforming growth factor β through expression of decoy receptors further demonstrates the extent to which effector T cells can be engineered for optimal anti-tumor activity (Foster AE et al., J Immunother. 2008; 31(5): 500-5). Thus, while it appears that CARs can trigger T cell activation in a manner similar to endogenous T cell receptors, the major obstacles to the clinical application of this technology have so far been the in vivo expansion of CAR+ T cells, the rapid disappearance of cells after infusion, and disappointing clinical activity. A variety of antibody-based modalities are currently being developed to target CD33-positive tumors, including anti-CD33 antibody-drug conjugates (Stein AS et al., Blood, 2015, 126(23), 324), bispecific T cell engagers (BiTEs) (Laszlo GS et al., Blood 2013: 123(4): 554-561), and CAR T cells (Wang QS et al., Mol Ther. 2015 Jan; 23(1): 184-91). Recent work in preclinical models of AML has shown that lysis of CD33-positive AML blasts and tumor cell lines can be achieved in vitro and in vivo with CD33-targeted modalities, but many challenges of this approach are becoming apparent in the clinical setting, including treatment-related toxicities (available at: www.businesswire.com / news / home / 20161227005087 / en / Seattle-Genetics-Announc es-Clinical-Hold-Phase-1; Rowe JM and B, Blood 2013 121:4838-4841, Wang QS et al., Mol Ther. 2015 Jan; 23(1):184-91, NCT01864902) and suboptimal efficacy (Walter RB et al., Blood. 2012; 119(26):6198-6208; Cowan AJ et al., Biosci 2013; 18(4):1311-1334). In addition, in BiTE-based approaches, the reliance on high-density CD33 antigen expression and the need for additional T cell co-stimulation / checkpoint blockade for optimal BiTE function remain challenges (Laszlo GS et al., Blood. 2014; 123(4):554-56, Laszlo GS et al., Blood Cancer Journal (2015) 5, e340). Therefore, there is an urgent and long-felt need in the art for new compositions and methods for treating AML using approaches that can exhibit specific and potent anti-tumor effects without the aforementioned drawbacks.

[0017] The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the present invention as disclosed and described herein provides CARs that can be used to treat diseases, disorders, or conditions associated with dysregulated CD33 expression, and the CARs comprise a CD33 antigen binding domain that exhibits high surface expression on transduced T cells, exhibits a high degree of cell lysis, and in vivo expansion and persistence of transduced T cells. Summary of the Invention

[0018] Provided herein are new anti-CD33 antibodies or their antigen binding domains and chimeric antigen receptors (CARs) comprising such CD33 antigen binding domains, as well as host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors. CAR can be composed of a single molecule expressed on the surface of effector cells, or CAR comprises a signal transduction module and a soluble targeting module expressed by effector cells, such as when a soluble targeting module is combined with a signal transduction module expressed by the cell, to form a complete functional CAR. CAR shows high surface expression on transduced T cells, with a high degree of cell lysis and amplification and persistence in vivo of transduced T cells. Also provided are methods using disclosed CARs, host cells, and nucleic acid molecules, such as for treating cancer in a subject.

[0019] Thus, in one aspect, an isolated polynucleotide encoding a human anti-CD33 antibody or fragment thereof is provided, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9 and 11.

[0020] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD33 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

[0021] In one embodiment, an isolated polynucleotide encoding a fully human anti-CD33 antibody or fragment thereof is provided, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

[0022] In one aspect, an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) is provided, wherein the chimeric antigen receptor (CAR) comprises, from N-terminus to C-terminus, at least one CD33 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the CD33 antigen binding domain is encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11.

[0023] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD33 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD33.

[0024] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD33 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD33.

[0025] In one embodiment, the targeting domain of CAR is independently expressed in the form of a monoclonal antibody, ScFv Fab, or Fab'2 and comprises an antigen targeting domain coupled to an additional binding tag or epitope, wherein the antigen targeting domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and the effector cell expression component of CAR comprises a binding domain specifically for binding to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0026] In one embodiment, the targeting domain of CAR is independently expressed in the form of a monoclonal antibody, ScFv Fab, or Fab'2 and comprises an antigen targeting domain and an additional scFv, wherein the antigen targeting domain comprises a nucleic acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and the effector cell expression component of CAR comprises a tag or epitope that specifically reacts with the additional scFv expressed on the soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0027] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD33 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) that binds to CD33.

[0028] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 antigen binding domain is connected to a transmembrane domain via a linker domain.

[0029] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD33 extracellular antigen binding domain is preceded by a sequence encoding a leader peptide or signal peptide.

[0030] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the CAR comprises at least one CD33 antigen binding domain, the CD33 antigen binding domain being encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and wherein the CAR further encodes an extracellular antigen binding domain that targets an antigen, including but not limited to CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0031] In certain embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the additionally encoded extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain. ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2 ScFv antigen binding domain, anti-NY-ESo-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0032] In one aspect, the CAR provided herein further comprises a linker or spacer domain.

[0033] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the extracellular CD33 antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

[0034] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28 and is connected to the transmembrane domain.

[0035] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0036] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0037] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

[0038] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0039] In other embodiments, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or a combination thereof.

[0040] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or signal peptide, wherein the leader or signal peptide nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43.

[0041] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

[0042] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD33 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0043] In one embodiment, a CAR is provided, wherein the extracellular CD33 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0044] In another embodiment, a CAR is provided, wherein at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0045] In some embodiments, a CAR is provided, wherein the CAR further encodes an extracellular antigen binding domain comprising CD19, CD20, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0046] In one embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain. ScFv antigen binding domain, an anti-GD-2 ScFv antigen binding domain, an anti-NY-ESo-1 TCR ScFv antigen binding domain, an anti-MAGE A3 TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0047] In another embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises only an immunoglobulin variable heavy chain (VH) anti-CD19 antigen binding domain, an anti-CD20 VH antigen binding domain, an anti-CD22 VH antigen binding domain, an anti-ROR1 VH antigen binding domain, an anti-mesothelin VH antigen binding domain, an anti-CD33 VH antigen binding domain, an anti-CD38 VH antigen binding domain, an anti-CD123 (IL3RA) VH antigen binding domain, an anti-CD138 VH antigen binding domain, an anti-BCMA (CD269) VH antigen binding domain, an anti-GPC2 VH antigen binding domain, an anti-GPC3 VH antigen binding domain, an anti-FGFR4 VH antigen binding domain, an anti-c-Met VH antigen binding domain, an anti-PMSA VH antigen binding domain, an anti-glycolipid F77 VH antigen binding domain, an anti-EGFRvIII VH antigen binding domain, an anti-GD-2 VH antigen binding domain, an anti-NY-ESO-1 TCR VH antigen binding domain, anti-MAGE A3 TCR VH antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0048] In another embodiment, a CAR is provided, wherein the extracellular antigen binding domain comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which can be derived from a natural or synthetic sequence comprising: an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-ROR1 P antigen binding domain, an anti-mesothelin P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSA P antigen binding domain, an anti-glycolipid F77 P antigen binding domain, an anti-EGFRvIII P antigen binding domain, an anti-GD-2 P antigen binding domain, an anti-NY-ESO-1 TCR β antigen binding domain, anti-MAGE A3 TCR β antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof. In another embodiment, a CAR is provided wherein at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0049] In another embodiment, a CAR is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0050] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 15 (LTG 1905EF1a VH-2 CD33-CD8 TM-41BB-CD3ζ nucleic acid sequence ( Figure 2A In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 16 (LTG 1905 EF1a VH-2 CD33-CD8 TM-41BB-CD3ζ amino acid sequence ( Figure 2A )).

[0051] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 17 (LTG1906 EF1a-VH-4 CD33-CD8 TM-41BB-CD3ζ nucleic acid sequence ( Figure 2B In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 18 (LTG 1906 EF1a-VH-4 CD33-CD8 TM-41BB-CD3ζ amino acid sequence ( Figure 2B )).

[0052] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (LTG1936 EF1a ScFv9 CD33 CD8 TM-41BB-CD3ζCAR nucleotide sequence ( Figure 2C In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 20 (LTG1936 EF1a ScFv9 CD33 CD8TM-41BB-CD3ζ CAR amino acid sequence ( Figure 2C )).

[0053] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 21 (LTG1937 EF1a ScFv10 CD33 CD8 TM-41BB-CD3ζ nucleic acid sequence ( Figure 2DIn one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 22 (LTG1937 EF1a ScFv10 CD33 CD8 TM-41BB-CD3 amino acid sequence ( Figure 2D )).

[0054] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 23 (LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3ζ nucleic acid sequence ( Figure 2E In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 24 (LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3ζ amino acid sequence ( Figure 2E )).

[0055] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 25 (LTG1939 EF1a_ScFv15 CD33 CD8 TM-41BB-CD3ζ nucleic acid sequence ( Figure 2F In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 26 (LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3ζ amino acid sequence ( Figure 2F )).

[0056] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 69 (LTG1927 EF1a-CD33_4 CD8 TM-CD28-CD3ζ nucleic acid sequence ( Figure 12A In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 70 (LTG1927 EF1a-CD33_4 CD8 TM-CD28-CD3ζ amino acid sequence ( Figure 12A )).

[0057] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 71 (LTG_D0033 Ef1a-CD33_4 VH TNFRSF19 H_TM_CD28z nucleic acid sequence ( Figure 12BIn one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 72 (LTG_D0033 (Ef1a-CD33_4 VH TNFRSF19H_TM_CD28z) amino acid sequence ( Figure 12B )).

[0058] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 73 (LTG_D0034 Ef1a-CD33_4 VH TNFRSF19 H_TM_4-1BBz nucleic acid sequence ( Figure 12C In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 74 (LTG_D0034 Ef1a-CD33_4 VH TNFRSF19H_TM_4-1BBz amino acid sequence ( Figure 12C )).

[0059] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 87 (LTG_D0035 Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z nucleic acid sequence ( Figure 12F In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 88 (LTG_D0035 Ef1a_CD33_4 VH HCH2 CH3 IgG4_CD8TM_CD28z amino acid sequence ( Figure 12F )).

[0060] In one aspect, the CARs disclosed herein are modified to express or comprise a detectable marker for use in diagnosing, monitoring and / or predicting treatment outcome (e.g., progression-free survival of a cancer patient) or for monitoring the progress of such treatment.

[0061] In one embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 75 (LTG_D0015 Ef1a-CD33_4 VH CD8 BBz T2A tEGFR nucleic acid sequence ( Figure 12D In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 76 (LTG_D0015 Ef1a-CD33_4 VH CD8 BBzT2A tEGFR amino acid sequence ( Figure 12D )).

[0062] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 77 (LTG_D0016 Ef1a-CD33_4 VH CD8 28z T2A tEGFR nucleic acid sequence ( Figure 12E In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 78 (LTG_D0015 Ef1a-CD33_4 VH CD8 28zT2A tEGFR amino acid sequence ( Figure 12E )).

[0063] In one embodiment, the nucleic acid molecule encoding the disclosed CAR can be contained in a vector, such as a viral vector. The vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

[0064] In certain embodiments, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0065] In another embodiment, the vector expressing CAR can also be modified to include one or more control CART cell expression or to eliminate CAR-T cell manipulation elements by means of a suicide switch. The suicide switch can include, for example, a drug that induces apoptosis-inducing signal transduction cascade or induces cell death. In a preferred embodiment, the vector expressing CAR can also be modified to express an enzyme, such as thymidine kinase (thymidine kinase, TK) or cytosine deaminase (cytosinedeaminase, CD).

[0066] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR is also provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0067] On the other hand, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signal transduction domain, and the extracellular antigen binding domain comprises a CD33 antigen binding domain comprising an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10 or 12, wherein the T cell is a T cell of a human suffering from cancer. The cancer particularly includes hematological cancers, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0068] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0069] In another embodiment, a pharmaceutical composition is provided wherein the human cancer comprises adult carcinoma, including: oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine corpus, ovary, vulva, vagina, prostate, testicle, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

[0070] In another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells from a human having cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), pediatric B-cell malignancies (including B-lineage ALL (acute lymphoblastic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0071] In another aspect, a method for preparing a T cell comprising a CAR (hereinafter referred to as a "CAR-T cell") is provided. The method comprises transducing a T cell with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds to CD33, thereby preparing a CAR-T cell.

[0072] In another aspect, a method for producing an RNA-engineered cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA encoding a nucleic acid molecule of the disclosed CAR into cells of a subject, thereby producing CAR cells.

[0073] In another aspect, a method for diagnosing a disease, disorder, or condition associated with CD33 expression on a cell is provided, comprising: a) contacting the cell with a human anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12; and b) detecting the presence of CD33, wherein the presence of CD33 is diagnostic for a disease, disorder, or condition associated with CD33 expression.

[0074] In one embodiment, the disease, disorder or condition associated with CD33 expression is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies including B-lineage ALL (acute lymphoblastic leukemia), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0075] In another embodiment, a method for diagnosing, prognosing, or determining risk of a CD33-associated disease in a mammal is provided, comprising detecting expression of CD33 in a sample derived from the mammal, comprising a) contacting the sample with a human anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12; and b) detecting the presence of CD33, wherein the presence of CD33 is diagnostic for the CD33-associated disease in the mammal.

[0076] In another embodiment, a method of inhibiting CD33-dependent T cell suppression is provided, comprising contacting a cell with a human anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In one embodiment, the cell is selected from the group consisting of a CD33-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0077] In another embodiment, a method is provided for blocking T cell suppression mediated by cells expressing CD33 and altering the tumor microenvironment to inhibit tumor growth in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In one embodiment, the cell is selected from a CD33-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0078] In another embodiment, a method is provided for inhibiting, suppressing, or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal, comprising administering to the mammal an effective amount of a composition comprising an isolated anti-CD33 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In one embodiment, the antibody or fragment thereof inhibits the interaction between a first cell and a T cell, wherein the first cell is selected from the group consisting of a CD33-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.

[0079] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding the disclosed CAR.

[0080] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more disclosed CARs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of a host cell expressing a disclosed CAR that specifically binds to CD33 and / or one or more of the foregoing antigens under the following conditions, the conditions being sufficient to form an immune complex of the antigen binding domain on the CAR with the extracellular domain of CD33 and / or one or more of the foregoing antigens in the subject.

[0081] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated expression of a tumor antigen is provided, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular CD33 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, the extracellular CD33 antigen binding domain comprising the amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10 or 12, or any combination thereof, and wherein the T cells are T cells of a subject suffering from cancer.

[0082] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cell comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one CD33 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the CD33 antigen binding domain comprises an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10 or 12, or any combination thereof, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the foregoing method, the at least one transmembrane domain comprises a transmembrane T cell receptor α, β or ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0083] In another embodiment, a method for generating a persistent population of genetically modified T cells in a human diagnosed with cancer is provided. In one embodiment, the method comprises administering to a human a T cell genetically modified to express a CAR, wherein the CAR comprises at least one CD33 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the CD33 antigen binding domain comprises an amino acid sequence of SEQ ID NO. 2, 4, 6, 8, 10, or 12, or any combination thereof, wherein after administration, the persistent population of genetically modified T cells or the progeny of the T cells persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years.

[0084] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0085] In aspects and embodiments of all methods described herein, any of the aforementioned cancers, diseases, disorders or conditions associated with elevated expression of tumor antigens can be treated, prevented or ameliorated using one or more CARs disclosed herein.

[0086] In another aspect, a kit for preparing a chimeric antigen receptor T cell as described above, or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression in a subject as described above, is provided, comprising a container and instructions for using the kit, wherein the container comprises any one of the nucleic acid molecules, vectors, host cells or compositions disclosed above, or any combination thereof.

[0087] It will be appreciated that CARs, host cells, nucleic acids, and methods are also applicable beyond the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description, which is made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 A schematic diagram depicting the general domain structure of a CAR with a novel extracellular CD33 antigen-binding domain sequence. The chimeric antigen receptor comprises an extracellular CD33-binding immunoglobulin single-chain variable fragment (ScFv) domain or only an immunoglobulin heavy chain variable fragment (VH) domain derived from CD8 ( Figure 1 A, B, F, G), TNFRSF19 ( Figure 1 C, D), IgG4 ( Figure 1 The hinge domain of E) is derived from CD8 ( Figure 1 A, B, E, F, G), TNFRSF19( Figure 1 The transmembrane domain of C and D is derived from CD137 / 4-1BB ( Figure 1 A, C, F) or CD28 ( Figure 1 Some bicistronic constructs incorporate a tag derived from truncated EGFR (tEGFR) via a ribosomal skipping 2A sequence ( Figure 1 F, G).

[0089] Figures 2A-2F A variety of chimeric antigen receptors (CARs) containing novel extracellular CD33 antigen-binding domain sequences are described. The general scheme of CAR includes a signal peptide from N-terminus to C-terminus, an anti-CD33 binder variable heavy chain fragment or a linked single-chain variable fragment (ScFv), an extracellular linker, a transmembrane, 4-1BB, and CD3ζ. Figure 2A Depicted are the lentiviral vector expressing the CAR, LTG 1905 EF1a VH-2 CD33-CD8 TM-41BB-CD3ζ nucleic acid sequence and the encoded amino acid sequence. Figure 2BDepicted is a CAR-expressing lentiviral vector comprising the LTG 1906 (EF1a-VH-4CD33-CD8 TM-41BB-CD3ζ) nucleic acid sequence and the encoded amino acid sequence. Figure 2C Depicted is a CAR-expressing lentiviral vector comprising the LTG1936 EF1a ScFv9 CD33CD8 TM-41BB-CD3ζ nucleotide sequence and the encoded amino acid sequence. Figure 2D Depicted is a CAR-expressing lentiviral vector comprising the LTG1937 EF1a ScFv10 CD33 CD8 TM-41BB-CD3ζ nucleic acid sequence and the encoded amino acid sequence. Figure 2E Depicted is a CAR-expressing lentiviral vector comprising the LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3ζ nucleic acid sequence and the encoded amino acid sequence. Figure 2F Depicted is a CAR-expressing lentiviral vector comprising the LTG1939 EF1aScFv15 CD33 CD8 TM-41BB-CD3ζ nucleic acid sequence and the encoded amino acid sequence.

[0090] Figure 3 Depicts anti-CD33 CART surface expression in primary human T cells. CAR T cells redirected to the CD33 tumor antigen using only the variable heavy chain targeting domain were generated by lentiviral transduction. CART detection was performed by flow cytometry. T cells were washed twice in cold PBS-EDTA buffer and stained with CD33-Fc peptide and then with anti-Fc-AF647 reagent. Data were acquired on a MACSQuant 10 flow cytometer in the APC channel. NT untransduced cells, GFP negative control.

[0091] Figure 4 Anti-CD33 CAR T cells incorporating immunoglobulin heavy chain variable domain binders are described, demonstrating cell lysis of CD33-positive tumors in vitro. CAR T cells expressing anti-CD33 constructs were incubated overnight with high CD33 (HL-60), medium CD33 (K562), and low CD33 (Reh) targets stably transduced with firefly luciferase at effector-target ratios of 5, 10, and 20. CART cytotoxic activity was then assessed by luciferase activity measurement as described in the Materials and Methods. N=3+ / -SEM.

[0092] Figure 5Depicts that VH-based CD33-specific CART cells produce high levels of cytokines when co-cultured with CD33-positive leukemia cell lines. Anti-CD33 CART cells were co-incubated with high CD33 (THP-1, HL-60), medium CD33 (K562), or low CD33 (Reh) leukemia cell lines at an E:T ratio of 10:1 overnight, and the supernatant was analyzed for cytokine concentrations by ELISA. N=3+ / -SD. Negative controls: NT untransduced T cells, 1398-GFP transduced T cells.

[0093] Figure 6 Depicts anti-CD33 CART surface expression in primary human T cells. CAR T cells redirected to the CD33 tumor antigen using a ScFv targeting domain were generated by lentiviral transduction. CAR T detection was performed by flow cytometry. T cells were washed twice in cold PBS-EDTA buffer and stained with CD33-Fc peptide and then with anti-Fc-AF647 reagent. Data were acquired on a MACSQuant 10 flow cytometer in the APC channel. UTD untransduced cells, 1398-GFP negative control.

[0094] Figure 7 Anti-CD33 CAR T cells incorporating immunoglobulin heavy chain variable domain binders are depicted, demonstrating cell lysis of CD33-positive tumors in vitro. CAR T cells expressing anti-CD33 constructs were incubated overnight with high CD33 (HL-60, MOLM-14), medium CD33 (K562), and low CD33 (Reh) targets stably transduced with firefly luciferase at effector-target ratios of 5, 10, and 20. CART cytotoxic activity was then assessed by luciferase activity measurement as described in Materials and Methods. N=3+ / -SEM.

[0095] Figure 8 Depicts that scFv-based and VH-based CD33-specific CAR T cells produce high levels of cytokines when co-cultured with CD33-positive leukemia cell lines. Anti-CD33 CART cells were co-incubated with high CD33 (HL-60, MOLM-14) or low CD33 (Reh) leukemia cell lines at a 1:1 E:T ratio overnight, and the supernatant was analyzed for cytokine concentrations by ELISA. N=3+ / -SD. Negative controls: UTD-untransduced T cells, 1398-GFP transduced T cells.

[0096] Figure 9 Depicts the interaction of CAR T cells expressing various anti-CD33 constructs with HL-60 CD33 +Long-term co-incubation assay of tumor cells. Anti-CD33 CAR T cell line was co-cultured with HL-60 CD33 + Tumor cells were combined in culture at the indicated effector-target (E:T) ratios and maintained for 11 days. Co-cultured cells were then harvested and analyzed by flow cytometry. Cells were gated based on singlet and side scatter peaks, and dead cells were excluded by 7-AAD staining, as described in Materials and Methods. Boxes indicate viable HL-60 cells per marker under each condition. + Tumor cells and CD3 + Percentage of CAR T cells. UTD = untransduced T cell control, 1398-GFP transduced T cell control, E:T 1:0 = T cell only control.

[0097] Figure 10 Figure 3 depicts the kinetics of tumor rejection by CD33-targeted CAR T cells assessed in vivo using bioluminescence imaging. NSG mice were inoculated with 1.0x10 6 MOLM-14CD33 + AML cells, and 5.0x10 6 CAR T + cells / mouse. Tumor burden was assessed weekly by bioluminescence imaging between days 14 and 35. A. Mean radiance + / - SEM, N = 6 mice / group. B. Kaplan-Meier curve depicting the percentage of mice in each experimental group that survived over the course of the experiment, N = 6 mice / group. TA - tumor only, UTD - untransduced T cell control.

[0098] Figure 11 The function of CD33-targeted CAR T cells evaluated in vivo is depicted. On day 0, NSG mice were inoculated with 1.0x10 6 MOLM-14 CD33 + AML cells, and 5.0x10 6 CAR T +Cells / mouse. Blood was collected from mice on day 19 of the study, and the levels of circulating CAR T, tumor cells, and inflammatory cytokines were analyzed. A. CART cells and MOLM-14 tumor cells were obtained by flow cytometry, and the absolute cell number was determined using CountBright beads. B. The levels of inflammatory cytokines in mouse plasma were assessed by MACS Human Multiplex Bead Array. N=6 mice / group. TA-tumor only, UTD-untransduced T cell control. Groups were compared by two-way ANOVA and Dunnett's post hoc test. ***p<0.001, *p<0.05, NS-not significant.

[0099] Figures 12A-12F Described in the case of different CAR configurations, a variety of chimeric antigen receptors (CARs) comprising new extracellular VH CD33_4 antigen binding domain sequences. The general scheme of CAR includes a signal peptide from N-terminal to C-terminal, an anti-CD33 binder variable heavy chain fragment extracellular joint, a transmembrane domain, a costimulatory domain, and a CD3ζ activation domain. Some sequences are included in the tEGFR tag peptide separated by a 2A ribosomal skipping sequence downstream of the CAR sequence. Figure 12A Depicted is a CAR-expressing lentiviral vector comprising the LTG1927 EF1a CD33_4 CD8™ CD28 CD3ζ nucleic acid sequence and the encoded amino acid sequence. Figure 12B Depicted is a CAR-expressing lentiviral vector comprising the LTG_D0033 EF1a CD33_4 VHTNFRSF19 H_TM_CD28ζ nucleic acid sequence and the encoded amino acid sequence. Figure 12C Depicted is a CAR-expressing lentiviral vector comprising the LTG_D0034 Ef1a_CD33_4 VH TNFRSF19 H_TM_4-1BB CD3ζ nucleic acid sequence and the encoded amino acid sequence. Figure 12D Depicted is a CAR-expressing lentiviral vector comprising the LTG_D0015 CD33_4VH CD8 BB CD3ζ T2AtEGFR nucleic acid sequence and the encoded amino acid sequence. Figure 12E Depicted is a CAR-expressing lentiviral vector comprising the LTG_D0016 CD33_4VH CD8 28 CD3ζ T2A tEGFR nucleic acid sequence and the encoded amino acid sequence. Figure 12F Depicted is a CAR-expressing lentiviral vector comprising the LTG_D0035 Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28CD3ζ nucleic acid sequence and the encoded amino acid sequence. DETAILED DESCRIPTION

[0100] definition

[0101] Unless the context clearly indicates otherwise, nouns used herein without quantifier modification refer to one / kind or more / kind. For example, the term "antigen" includes one or more antigens and can be considered to be equivalent to the phrase "at least one antigen". The term "comprising" used herein means "including". Thus, "comprising an antigen" means "including an antigen" without excluding other elements. The phrase "and / or" means "and" or "or". It should also be understood that, unless otherwise indicated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular mass values are approximate and are provided for descriptive purposes. Although many methods and materials similar or equivalent to the methods and materials described herein can be used, specific suitable methods and materials are described below. In the event of a conflict, this specification (including the explanation of the terms) will prevail. In addition, materials, methods and examples are only illustrative and are not intended to be limiting. For the ease of the review of different embodiments, the following term explanations are provided:

[0102] The term "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to encompass variations of +-.20%, or in some cases +-.10%, or in some cases +-.5%, or in some cases +-.1%, or in some cases +-.0.1% from the particular value, as such variations are suitable for performing the disclosed methods.

[0103] Unless otherwise indicated, technical terms herein are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0104] The present disclosure provides CD33 antibodies or fragments thereof and chimeric antigen receptors (CARs) with such CD33 antigen binding domains. The functional activity enhancement of CAR is directly related to the functional activity enhancement of CAR-expressing T cells. As a result of one or more of these modifications, CAR exhibits both high cytokine-induced lysis and cell surface expression on transduced T cells, as well as increased in vivo T cell amplification levels and the persistence of transduced CAR-expressing T cells.

[0105] The unique ability to combine functional parts derived from different protein domains is a key innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains is a key design feature, as is the way in which its specificity is combined. Each design domain is an essential component that can be used between different CAR platforms to transform the function of lymphocytes. For example, the selection of extracellular binding domains can make otherwise ineffective CARs effective.

[0106] The constant framework components of the immunoglobulin-derived protein sequences used to establish the extracellular antigen-binding domains of CARs can be completely neutral or they can self-associate and drive T cells to a state of metabolic exhaustion, thereby making the effect of the therapeutic T cells expressing the CAR much lower. This occurs independently of the antigen binding function of the CAR domain. In addition, the selection of intracellular signal transduction domains can also control the activity and persistence of therapeutic lymphocyte populations for immunotherapy. Although the ability to bind target antigens and transmit activation signals to T cells through these extracellular and intracellular domains, respectively, is an important aspect of CAR design, it has also become apparent that the choice of extracellular antigen-binding fragment source can have a significant effect on the efficacy of CAR, thereby having a limiting effect on the function and clinical utility of CAR.

[0107] Surprisingly and unexpectedly, it has now been found that the use of fully human antigen-binding domains in CARs, rather than mouse-derived antigen-binding fragments that are prone to inducing anti-mouse immune responses and CAR T elimination in the host (see, the UPenn-sponsored clinical trial using mouse-derived SS1 ScFv sequence, NCT02159716), can also determine the functional activity of CAR-expressing T cells.

[0108] The CAR disclosed herein is expressed at high levels in cells. Cells expressing CAR have a high in vivo proliferation rate, produce a large amount of cytokines, and have high cytotoxic activity against cells with CD33 antigens bound to CAR on the surface. The use of human extracellular CD33 antigen binding domains results in the production of CARs that work better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response, and killing CAR T cell populations. CARs expressing the entire human extracellular CD33 ScFv antigen binding domain exhibit excellent activity / characteristics, including: i) preventing poor CAR T persistence and function as seen with mouse-derived binding sequences; ii) enabling effective delivery to areas lacking CAR (i.e., intrapleural); and iii) the ability to produce CAR T cell designs based on binders with both high and low affinity for CD33. This latter property allows researchers to better tune the efficacy versus toxicity and / or tissue specificity of CAR T products because lower affinity binders can have higher specificity for tumors compared to normal tissues due to higher CD33 expression on tumors compared to normal tissues, which can prevent on-target off tumor toxicity and bystander cell killing.

[0109] The following is a detailed description of the CAR of the present invention, including a description of its extracellular CD33 antigen binding domain, transmembrane domain, and intracellular domain, as well as additional descriptions of CAR, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors and host cells, treatment methods, compositions, and kits for using the disclosed CAR.

[0110] A. Chimeric Antigen Receptor (CAR)

[0111] The CAR disclosed herein comprises at least one CD33 antigen binding domain capable of binding to CD33, at least one transmembrane domain, and at least one intracellular domain.

[0112] Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide comprising an antigen binding domain (e.g., single-chain variable fragment (ScFv)) of an antibody connected via a transmembrane domain to a T cell signaling domain. The characteristics of CAR include its ability to redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner, and the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0113] As disclosed herein, the intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a CAR that includes an intracellular domain of a T cell receptor, such as, but not limited to, an intracellular portion of a CD3ζ protein. A costimulatory signaling domain refers to a portion of a CAR that includes an intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of a lymphocyte to an antigen.

[0114] 1. Extracellular domain

[0115] In one embodiment, CAR comprises a target-specific binding element, which is also referred to as an antigen binding domain or portion. The selection of domains depends on the type and quantity of the ligands that define the surface of the target cell. For example, an antigen binding domain can be selected to identify a ligand that acts as a cell surface marker associated with a specific disease state on the target cell. Therefore, some examples of cell surface markers that can serve as ligands for antigen binding domains in CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0116] In one embodiment, CAR can be modified to target a tumor antigen of interest by modifying a desired antigen binding domain that specifically binds to an antigen on a tumor cell. Tumor antigens are proteins produced by tumor cells that trigger an immune response, particularly a T cell-mediated immune response. The choice of antigen binding domain will depend on the specific type of cancer to be treated. Tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD33. The tumor antigens disclosed herein are included by way of example only. This list is not meant to be exclusive and additional examples will be apparent to those skilled in the art.

[0117] In one embodiment, tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumor. Malignant tumor expresses a variety of proteins that can be used as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecule group, such as oncogene HER-2 / Neu / ErbB-2. Another group of target antigens is cancer-embryonic antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotypic immunoglobulin constitutes the unique true tumor-specific immunoglobulin antigen of individual tumor. B cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypic) have been used as targets for passive immunotherapy using monoclonal antibodies, but with limited success.

[0118] In a preferred embodiment, the tumor antigen is CD33, and tumors associated with CD33 expression include lung cancer mesothelioma, ovarian cancer, and pancreatic cancer, or any combination thereof, which express high levels of the extracellular protein CD33.

[0119] Types of tumor antigens can also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or TAAs can be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0120] Non-limiting examples of TSAs or TAAs include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0121] In one embodiment, the antigen binding domain portion of the CAR targets antigens including but not limited to: CD19, CD20, CD22, ROR1, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGEA3 TCR, etc.

[0122] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD33 antigen.

[0123] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 VH-2 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 1, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 VH-2 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0124] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 VH-4 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 VH-4 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0125] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv 9 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 5, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 ScFv 9 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0126] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv 10 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 7, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 ScFv 10 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0127] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv 12 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 9, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 ScFv 12 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0128] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD33 ScFv 15 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 11, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 ScFv 15 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0129] The generation and binding characteristics of the specific CD33 variable heavy chain only and ScFv antigen binding fragments or antigen binders described herein are shown in Example 1.

[0130] In various embodiments of the CD33-specific CAR disclosed herein, the general scheme is Figure 1 , and comprises, from N-terminus to C-terminus, a signal or leader peptide, anti-CD33 ScFv, an extracellular linker, CD8 transmembrane, 4-1BB, CD3ζ, wherein boldface letters indicate cloning sites for linking domains.

[0131] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 16 [LTG 1905 EF1a VH-2 CD33-CD8 TM-41BB-CD3ζ amino acid sequence (e.g. Figure 2A shown)].

[0132] In one embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 15, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 16, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof [LTG 1905 EF1a VH-2 CD33-CD8 TM-41BB-CD3ζ amino acid sequence (e.g., Figure 2A shown)].

[0133] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 17, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 18 [LTG 1906 EF1a-VH-4 CD33-CD8 TM-41BB-CD3ζ amino acid sequence (e.g. Figure 2B shown)].

[0134] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 18, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof [LTG 1906 EF1a-VH-4 CD33-CD8 TM-41BB-CD3ζ amino acid sequence (e.g., Figure 2B shown)].

[0135] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 19, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 20 [LTG1936 EF1a ScFv9 CD33 CD8 TM-41BB-CD3ζCAR amino acid sequence (e.g. Figure 2C shown)].

[0136] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 19, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 20, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof [LTG1936 EF1a ScFv9 CD33 CD8 TM-41BB-CD3ζCAR amino acid sequence (e.g., Figure 2C shown)].

[0137] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 21, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 22 [LTG1937 EF1a ScFv10 CD33 CD8 TM-41BB-CD3 amino acid sequence (e.g. Figure 2D shown)].

[0138] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 21, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof [LTG1937 EF1a ScFv10 CD33 CD8 TM-41BB-CD3 amino acid sequence (e.g., Figure 2D shown)].

[0139] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 23, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 24 [LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3ζ amino acid sequence (e.g. Figure 2E shown)].

[0140] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 23, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 24, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof [LTG1938 EF1a ScFv12 CD33 CD8 TM-41BB-CD3ζ amino acid sequence (e.g., Figure 2E shown)].

[0141] In another embodiment, the nucleic acid sequence encoding CAR comprises the nucleic acid sequence of SEQ ID NO: 25, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 26 [(LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3ζ amino acid sequence (such as Figure 2F shown)].

[0142] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 25, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 26, or a sequence 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto [(LTG1939 EF1a ScFv15 CD33 CD8 TM-41BB-CD3ζ amino acid sequence (e.g. Figure 2F shown)].

[0143] Surface expression of anti-CD33 CARs incorporating immunoglobulin heavy chain variable domain (VH) and single chain variable fragment (ScFv) sequences reactive with the CD33 antigen is shown in Example 2 below and summarized in Table 2. The expression level of each CAR containing ScFv or VH was determined by flow cytometric analysis of LV-transduced T cells from healthy donors using recombinant CD33-Fc peptide followed by an anti-human Fc F(ab')2 fragment conjugated to AF647 and detected in the APC channel (see Example 2, Table 2). Figure 3 and 6 ). VH-based anti-CD33CAR constructs 1905 and 1906 (black traces) were readily detected on the surface of T cells from both donors, demonstrating the reproducibility of T cell transduction. In contrast, no CAR expression was detected in negative control untransduced T cells (grey traces) and GFP controls (not shown), thus demonstrating the specificity of the detection method used (see Example 2, Figure 3 and Table 2). Similarly, ScFv-based anti-CD33 CAR constructs 1936, 1937, 1938, and 1939 were highly expressed in human primary T cells (black traces) compared to untransduced T cell controls (grey traces). Representative results from one donor are shown.

[0144] As in Example 2 Figure 4 and Figure 7As shown, high cytolytic activity of CD33 CARs was demonstrated when lentiviral vectors (LVs) expressing the following CARs were generated and tested for their anti-leukemic activity. Each experimental CAR contained the 4-1BB / CD3-ζ chain signaling motif and a specific anti-CD33 binding motif / domain as indicated herein. Four leukemic target cell lines with different CD33 surface expression were used: HL-60 and MOLM-14 (high), Reh and K562 (low). The VH domain-based CAR-T constructs LTG1905 and LTG1906 lysed low CD33 K562 cells, with LTG1906 showing excellent cytolytic function at the effector to target (E:T) ratios listed on the x-axis (see Figure 4 , LTG1905, and LTG1906, black diamonds and circles, respectively). When used in combination with the high CD33 HL-60 tumor cell line, LTG1906, but not LTG1905, demonstrated potent cytolytic function, highlighting the robustness of the construct LTG1906. In contrast, neither of the negative control groups, NT (untransduced T cells) nor 1398 (T cells transduced with a GFP control), exerted specific cytolytic activity. Therefore, the cytolytic activity of the anti-CD33 CARs LTG1906 and LTG1905 against CD33-expressing tumor cell lines we observed was both target-specific and CAR T-dependent.

[0145] In contrast, the ScFv-based anti-CD33 CAR constructs LTG1936 and LTG1939 were able to effectively lyse the high CD33 tumor cell lines HL-60 and MOLM-14, whereas they only partially lysed the low CD33 Reh tumor cell line and had no specific lytic activity against K562 (see Figure 7 , LTG1398 and LTG1936, white squares and white inverted triangles, respectively). This finding demonstrates the efficiency and specificity of the generated CAR constructs. Unexpectedly, the other CAR constructs tested in this group, LTG1937 and LTG1938, were inefficient at lysing high CD33 tumor cell lines, thus demonstrating again that CART design is not trivial and that soluble antibody features cannot be directly translated into CAR function.

[0146] The cytokine secretion capacity of anti-CD33 CAR T cells was then assessed. Tumor cells were co-incubated with CAR T cells or control T cells at an effector to target ratio of 10:1 overnight, and the culture supernatants were analyzed for IFNγ, TNFα, and IL-2 by ELISA (see Figure 5and Table 2). Notably, CAR T-expressing cells LTG1905 and LTG1906 produced high levels of IFNγ, TNFα, and IL-2, while the negative control NT and 1398 groups did not produce detectable cytokine induction. Unexpectedly, CD33 CAR LTG1905 tended to produce higher levels of induced cytokines against all tested tumor cell lines compared to construct LTG1906. This result is consistent with the lower in vitro cell lysis function of LTG1905 compared to LTG1906 (see Figure 4 ), instead, indicating the need to test multiple CAR T functional endpoints on a construct-by-construct basis.

[0147] Without intending to be limited to any particular mechanism of action, it is believed that possible reasons for the enhanced therapeutic function associated with exemplary CARs of the present invention include, for example, but are not limited to: a) improved lateral movement within the plasma membrane, thereby allowing more efficient signal transduction; b) superior localization within plasma membrane microdomains (e.g., lipid rafts) and greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior localization within the plasma membrane by preferentially moving away from inhibitory or downregulatory interactions (e.g., lower proximity or interaction with phosphatases such as CD45); and d) superior assembly into T cell receptor signaling complexes (i.e., immunological synapses); or any combination thereof.

[0148] Although the present disclosure has been illustrated using exemplary extracellular CD33 variable heavy chain-only and ScFv antigen binding domains, other nucleotide and / or amino acid variants within the CD33 variable heavy chain-only and ScFv antigen binding domains can be used to derive CD33 antigen binding domains for use in the CARs described herein.

[0149] Depending on the desired antigen to be targeted, the CAR can be further engineered to include a suitable antigen binding domain specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as an antigen binding domain incorporated into the CAR.

[0150] In an exemplary embodiment, the antigen binding domain portion of the CAR also targets CD19. Preferably, the antigen binding domain in the CAR is an anti-CD19 ScFV, wherein the nucleic acid sequence of the anti-CD19 ScFV comprises the sequence shown in SEQ ID NO: 37. In one embodiment, the anti-CD19 ScFV comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the anti-CD19 ScFV portion of the CAR comprises the amino acid sequence shown in SEQ ID NO: 38.

[0151] In one aspect of the present invention, a CAR is provided that is capable of binding to a non-TSA or non-TAA, including, for example, but not limited to, antigens derived from Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), Rubella virus, Coronavirus, Vesicular Stomatitis Virus, Rabies virus, Ebola virus, Parainfluenza virus, Mumps virus, Measles virus, Respiratory Syncytial Virus, Influenza virus, Hepatitis B virus, Parvovirus, Adenoviridae, Herpesviridae [e.g., Herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus], Poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or Hepatitis C virus, or any combination thereof.

[0152] In another aspect of the present invention, a CAR is provided that is capable of binding to an antigen derived from the following bacterial strains: Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, CARs are provided that can bind to antigens derived from infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, bacterial strains of the genus Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus, and the like. Streptococcus) (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or a combination thereof.

[0153] 2. Transmembrane domain

[0154] With respect to the transmembrane domain, the CAR comprises one or more transmembrane domains fused to the extracellular CD33 antigen binding domain of the CAR.

[0155] The transmembrane domain may be derived from a natural source or a synthetic source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0156] The membrane-spanning region used particularly in CAR described herein can be derived from the following (i.e., comprising at least the following membrane-spanning region): α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the membrane-spanning domain can be synthetic, in which case it will mainly comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic membrane-spanning domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection between the membrane-spanning domain and the cytoplasmic signal transduction domain of CAR. Glycine-serine doublets provide particularly suitable linkers.

[0157] In one embodiment, in addition to the transmembrane domains described above, a transmembrane domain that is naturally associated with one of the domains in the CAR is used.

[0158] In some cases, the transmembrane domain can be selected by amino acid substitution to prevent such domain from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0159] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 28.

[0160] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 28, or a sequence with 95 to 99% identity to an amino acid sequence of SEQ ID NO: 28.

[0161] In some cases, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 30, or a sequence with 95 to 99% identity thereto.

[0162] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0163] In one embodiment, the transmembrane domain in the CAR of the present invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 52. In another embodiment, the TNFRSF19 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 52.

[0164] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 52, or a sequence with 95 to 99% identity to an amino acid sequence of SEQ ID NO: 52.

[0165] 3. Spacer domain

[0166] In CAR, the spacer domain (also referred to as hinge domain) can be arranged between the extracellular domain and the membrane-spanning domain, or between the intracellular domain and the membrane-spanning domain. The spacer domain means any oligopeptide or polypeptide for connecting the membrane-spanning domain to the extracellular domain and / or the membrane-spanning domain to the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0167] In some embodiments, the linker may comprise a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill in the art and include those listed in U.S. Patent Nos. 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5 , 530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0168] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signal transduction into the cell. Some examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0169] As the spacer domain, all or part of amino acids 118 to 178 of the hinge region of CD8α (SEQ ID NO: 31) (NCBI RefSeq: NP.sub.-001759.3), amino acids 135 to 195 of CD8β (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP.sub.-000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP.sub.-006130.1) can be used. Alternatively, a portion of the constant region of the antibody H chain or L chain (CH1 region or CL region, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 32) can be used as the spacer domain. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0170] In addition, all or part of the amino acid sequence of the constant region of human IgG4 (UniProt ID: P01861) comprising CH1 (amino acids 1 to 98), hinge (SEQ ID NO: 80, and corresponding nucleotides SEQ ID NO: 79) (amino acids 99 to 110), CH2 (amino acids SEQ ID NO: 81 and corresponding nucleotides SEQ ID NO: 80) (amino acids 111 to 220), and CH3 (SEQ ID NO: 84, and corresponding nucleotides SEQ ID NO: 83) (amino acids 221 to 327), or a combination thereof, such as the IgG4 hinge CH2 CH3 domain (SEQ ID NO: 86 and corresponding nucleotides SEQ ID NO: 85), may be used.

[0171] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising the nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises the amino acid sequence of SEQ ID NO: 54, or a sequence with 95 to 99% identity thereof.

[0172] In one embodiment, the spacer domain of the CAR comprises a truncated hinge domain of TNFRSF19 comprising the nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the truncated hinge domain of TNFRSF19 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56. In another embodiment, the truncated hinge domain of TNFRSF19 comprises the amino acid sequence of SEQ ID NO: 56, or a sequence with 95 to 99% identity thereof.

[0173] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence with 95 to 99% identity thereof.

[0174] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 58, or a sequence with 95 to 99% identity thereof.

[0175] In addition, in CAR, a signal peptide sequence (also called a leader peptide) can be connected to the N-terminus. The signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins and is 15 to 30 amino acids in length. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for CAR. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 14.

[0176] In one embodiment, the CD8α leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8α leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44, or a sequence with 95 to 99% identity thereof.

[0177] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, or a sequence with 95 to 99% identity thereof.

[0178] In another embodiment, the TNFRSF19 leader peptide comprises the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the TNFRSF19 leader peptide and the CD8α leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8α hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, or a sequence with 95 to 99% identity thereof.

[0179] In one embodiment, the tag sequence encoding a truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the tEGFR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence with 95 to 99% identity thereto.

[0180] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 66 or a sequence having 95 to 99% identity thereto.

[0181] In one embodiment, the upstream furin recognition site and T2A self-cleaving peptide sequence and the furin recognition downstream site designed for simultaneous bicistronic expression of the tag sequence and the CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68 or a sequence having 95 to 99% identity thereto.

[0182] In one embodiment, the targeting domain of CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, or Fab'2 and is contained in a binding tag or epitope, while the component expressed by the effector cell of CAR comprises a binding domain specifically for a tag or epitope expressed on a soluble CAR module, for example, the specific binding of the soluble component of CAR to the cell binding component forms a complete functional CAR structure.

[0183] 4. Intracellular domain

[0184] The cytoplasmic domain of CAR or another intracellular signal transduction domain is responsible for activating at least one normal effector function of the immune cell in which CAR has been placed. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cell lysis activity or auxiliary activity, including secretion of cytokines. Therefore, the term "intracellular signal transduction domain" refers to a protein portion that transduces effector function signals and instructs cells to perform specialized functions. Although the entire intracellular signal transduction domain can usually be used, in many cases it is not necessary to use the entire chain. In terms of using a truncated portion of an intracellular signal transduction domain, such a truncated portion can be used to replace the complete chain, as long as it transduces the effector function signal. The term intracellular signal transduction domain therefore means any truncated portion that is sufficient to transduce the effector function signal including the intracellular signal transduction domain.

[0185] Some preferred examples of intracellular signaling domains for CARs include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same functional capability.

[0186] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or costimulatory signals are also required. Therefore, T cell activation can be considered to be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0187] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0188] Some examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful for the CARs disclosed herein include those derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3ζ (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of FcεRIγ (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of FcεRIβ (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3γ (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3δ (NCBI RefSeq: NP.sub.--000723.1), amino acids 153 to 207 of CD3ε (NCBI RefSeq: NP.sub.--000724.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--000136.1), amino acids 137 to 180 of CD3γ (NCBI RefSeq: NP.sub.--000064.1), amino acids 140 to 159 of CD3δ (NCBI RefSeq: NP.sub.--000724.1), amino acids 151 to 180 of CD3ε (NCBI RefSeq: NP.sub.--000725.1), amino acids 153 to 209 of CD3ε (NCBI RefSeq: NP.sub.--000726.1), amino acids 154 to 180 of CD3γ (NCBI RefSeq: NP.sub.--000136.1), amino acids 155 to 180 of CD3ε (NCBI RefSeq: RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of CD79a (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1) and amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), and variants thereof having the same function as these peptides. The amino acid numbering based on the amino acid sequence information of NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor of each protein (including signal peptide sequence, etc.). In one embodiment, the cytoplasmic signal transduction molecule in the CAR comprises a cytoplasmic signal transduction sequence derived from CD3ζ.

[0189] In a preferred embodiment, the intracellular domain of CAR can be designed to include a CD3-ζ signal transduction domain alone or in combination with any other desired cytoplasmic domain that can be used for CAR background. For example, the intracellular domain of CAR can include CD3ζ chain portion and costimulatory signal transduction region. Costimulatory signal transduction region refers to the part of the intracellular domain comprising costimulatory molecules in CAR. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Some examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands specifically bound to CD83, etc. Specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8α (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBI RefSeq: NP.sub.--001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), as well as variants thereof having the same functions as these peptides. Therefore, although the disclosure herein is primarily exemplified by 4-1BB as a costimulatory signal transduction element, other costimulatory elements are also within the scope of the present disclosure.

[0190] The cytoplasmic signal transduction sequences within the cytoplasmic signal transduction portion of CAR can be connected to each other in a random or specific order. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0191] In one embodiment, the intracellular domain is designed to include the signal transduction domain of CD3-ζ and the signal transduction domain of CD28. In another embodiment, the intracellular domain is designed to include the signal transduction domain of CD3-ζ and the signal transduction domain of 4-1BB. In another embodiment, the intracellular domain is designed to include the signal transduction domain of CD3-ζ and the signal transduction domain of CD28 and 4-1BB.

[0192] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 45 or SEQ ID NO: 59, respectively, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 47 or SEQ ID NO: 61, respectively.

[0193] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0194] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB comprises the amino acid sequence shown in SEQ ID NO: 34, SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48 or SEQ ID NO: 62, respectively.

[0195] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises the nucleic acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 59, respectively, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 47 or SEQ ID NO: 61, respectively.

[0196] In one embodiment, the intracellular domain in the CAR is designed to comprise the signal transduction domain of CD28 and the signal transduction domain of CD3-ζ, wherein the signal transduction domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signal transduction domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0197] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises the amino acid sequence shown in SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48 or SEQ ID NO: 62, respectively.

[0198] 5. Additional description of CAR

[0199] Also explicitly included within the scope of the present invention are the functional parts of CAR disclosed herein.When used with reference to CAR, the term "functional part" refers to any of the following parts or fragments of one or more CAR disclosed herein, and the part or fragment retains the biological activity of CAR (the part or fragment being a part thereof) (parent CAR). Functional part covers the following parts of, for example, CAR, which retains the ability to identify target cells or detect, treat or prevent diseases to a similar extent, to the same extent or to a higher degree compared to parent CAR. With reference to parent CAR, functional part can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of parent CAR.

[0200] The functional portion may comprise additional amino acids at the amino or carboxyl terminus or at both ends of the portion that are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance biological activity compared to the biological activity of the parent CAR.

[0201] Included within the scope of the present disclosure are functional variants of CAR disclosed herein. The term "functional variant" as used herein refers to a CAR, polypeptide or protein having obvious or significant sequence identity or similarity to a parent CAR, and the functional variant retains the biological activity of CAR (the functional variant being its variant). Functional variants encompass the following variants of, for example, CAR (parent CAR) described herein, which retain the ability to identify target cells to a similar extent, to the same extent or to a higher degree than the parent CAR. With reference to parent CAR, functional variants can, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or higher identity in terms of amino acid sequence with parent CAR.

[0202] Functional variants may, for example, comprise the amino acid sequence of a parent CAR having at least one conservative amino acid substitution. Alternatively or in addition, functional variants may comprise the amino acid sequence of a parent CAR having at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is improved compared to the parent CAR.

[0203] The amino acid substitutions of the CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with specific physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substitutions of an amino acid with a β-branched side chain for another amino acid with a β-branched side chain (e.g., He, Thr and Val), substitutions of an amino acid with an aromatic side chain for another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

[0204] The CAR can consist essentially of one or more specific amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0205] CAR (including functional portions and functional variants) can be of any length, i.e., can comprise any number of amino acids, as long as the CAR (or its functional portion or functional variant) retains its biological activity, such as the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal. For example, the CAR can be from about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0206] CAR (including functional parts and functional variants of the present invention) can include synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, di ... Indole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0207] CARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts and / or optionally dimerized or multimerized, or conjugated.

[0208] CAR (including its functional part and functional variant) can be obtained by methods known in the art. CAR can be prepared by any suitable method for preparing polypeptides or proteins. Suitable methods for synthesizing polypeptides and proteins from scratch are described in, for example, the following references: Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. In addition, polypeptides and proteins can be recombinantly produced using standard recombinant methods using the nucleic acids described herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In addition, some CARs (including functional portions and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Methods of separation and purification are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by a company. In this regard, CAR can be synthetic, recombinant, isolated and / or purified.

[0209] B. Antibodies and Antigen-Binding Fragments

[0210] One embodiment also provides a CAR that specifically binds to one or more antigens disclosed herein, a T cell expressing CAR, an antibody, or an antigen binding domain or portion thereof. As used herein, "CAR-expressing T cell" or "CAR T cell" means a T cell that expresses CAR and has an antigen specificity determined by, for example, the antibody-derived targeting domain of the CAR.

[0211] As used herein, "antigen binding domain" may include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, complete immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen.

[0212] "Monoclonal antibody" is an antibody obtained from a group of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations that may exist in small amounts, the individual antibodies constituting the group are identical. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of antibodies by any particular method. In some instances, a monoclonal antibody is an antibody produced by a single clone of a B lymphocyte or by a cell or its progeny that has been transfected with nucleic acids encoding a single antibody light chain variable region and a heavy chain variable region (or its antigen-binding fragment). In some instances, the monoclonal antibody is separated from the subject. The monoclonal antibody can have a conservative amino acid substitution that has substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, referring to Harlow & Lane, Antibodies, A Laboratory Manual, Second Edition. Cold Spring Harbor Publications, New York (2013).

[0213] Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0214] Each heavy and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). In some embodiments, the heavy and light chain variable regions combine to specifically bind to an antigen. In other embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies composed of only heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363: 446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3: 733-736, 1996). Reference to "VH" or "VH" refers to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment such as Fv, ScFv, dsFv, or Fab. References to "VL" or "VL" refer to the variable domain of an antibody light chain, including the variable domain of an Fv, ScFv, dsFv, or Fab.

[0215] The light and heavy chain variable regions comprise a "framework" region interrupted by three hypervariable regions also called "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework regions of the constituent light and heavy chains) serves to position and align the CDRs in three-dimensional space.

[0216] The CDRs are primarily responsible for binding to antigenic epitopes. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," Fifth Edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27: 55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also typically identified by the chain in which the specific CDR is located. Thus, a VH CDR3 is the CDR3 of the heavy chain variable domain of the antibody in which it is found, and a VL CDR1 is the CDR1 of the light chain variable domain of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0217] "Antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize a cognate antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of whole antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vol. 1-2, 2nd ed., Springer Press, 2010).

[0218] Single-chain antibodies (ScFv) are genetically engineered molecules comprising the VH and VL domains of one or more antibodies connected as a genetically fused single-chain molecule by a suitable polypeptide linker (see, e.g., Bird et al., Science, 242: 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry, ID Rugs, 13: 543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is generally not critical for ScFv. Therefore, ScFvs with two possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0219] In dsFv, the heavy and light variable chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90: 6444-6448, 1993; Poljak et al., Structure, 2: 1121-1123, 1994).

[0220] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0221] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy and variable light chains as described in Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference. These and other methods for preparing, for example, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, single-chain antibodies, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14: 243-246 (1993); Ward et al., Nature 341: 544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0222] An "antibody that binds to the same epitope as a reference antibody" is one that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, or vice versa, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0223] "Humanized" antibodies or antigen binding fragments comprise human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) antibodies or antigen binding fragments. The non-human antibody or antigen binding fragment providing CDR is referred to as a "donor," and the human antibody or antigen binding fragment providing the framework is referred to as a "recipient." In one embodiment, all CDRs in a humanized immunoglobulin are from a donor immunoglobulin. The constant region does not need to exist, but if it exists, it can be substantially identical to a human immunoglobulin constant region, e.g., an identity of at least about 85% to 90%, e.g., about 95% or higher. Therefore, all parts of a humanized antibody or antigen binding fragment (except possibly CDR) are substantially identical to corresponding parts of a natural human antibody sequence.

[0224] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies, which are generally of different species. In some instances, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0225] A "fully human antibody" or "human antibody" is an antibody that comprises a sequence from (or derived from) the human genome and does not comprise a sequence from another species. In some embodiments, a human antibody comprises CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for producing antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manuel. First edition New York: Cold Spring Harbor Laboratory Press, 2004. Print; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0226] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, and bispecific or bifunctional antibodies have two different binding sites.

[0227] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., supra, U.S. Patent Application Publication No. 2002 / 0197266 A1 and U.S. Patent No. 7,338,929).

[0228] Additionally, the CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof can be modified to include a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle).

[0229] C. Conjugates

[0230] CARs, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments thereof that are specific for one or more antigens disclosed herein can be conjugated to reagents such as effector molecules or detectable markers using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which effector molecules or detectable markers are covalently attached to antibodies or antigen-binding fragments that specifically bind to one or more antigens disclosed herein. It will be appreciated by those skilled in the art that a variety of effector molecules and detectable markers can be used, including but not limited to chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g., 125 I. 32 P. 14 C. 3 H and 35 S) and other labels, target moieties and ligands, etc.

[0231] The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin that is used to cause the death of a particular target cell (e.g., a tumor cell).

[0232] The operation for connecting an effector molecule or a detectable marker to an antibody or Fab changes according to the chemical structure of the effector. Polypeptides generally contain a variety of functional groups that can be used to react with suitable functional groups on the antibody to bind an effector molecule or a detectable marker, such as carboxylic acid groups (COOH), free amine groups (-NH2) or sulfhydryl groups (-SH). Alternatively, the antibody or Fab is derivatized to expose or connect other reactive functional groups. Derivatization can involve connecting any of a variety of known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. A linker can be any molecule for connecting an antibody or Fab to an effector molecule or a detectable marker. A linker can form a covalent bond with the antibody or Fab and with the effector molecule or a detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be attached to the constituent amino acids via its side groups (e.g., via a disulfide bond to cysteine) or to the alpha-carbon amino and carboxyl groups of the terminal amino acid.

[0233] In several embodiments, the linker can comprise a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill in the art and include those listed in U.S. Patent Nos. 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5 , 530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0234] In some embodiments, the joint is cleavable under intracellular conditions so that the cutting of the joint in the intracellular environment releases effector molecules or detectable markers from the antibody or Fab. In other embodiments, the joint is non-cleavable and the effector molecules or detectable markers are released, for example, by antibody degradation. In some embodiments, the joint can be cut by a cutting agent present in the intracellular environment (for example, in a lysosome or endosome or caveolae). The joint can be a peptide joint that is cut, for example, by an intracellular peptidase or protease (including but not limited to a lysosome or endosomal protease). In some embodiments, the peptide joint is at least 2 amino acids long or at least 3 amino acids long. However, the joint can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, 1 to 15 amino acids long. Proteases can include cathepsin B and cathepsin D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives such that the active drug is released within the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptide linker cleavable by the sulfhydryl-dependent protease cathepsin-B (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker) can be used. Other examples of such linkers are described, e.g., in U.S. Patent No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0235] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable under neutral pH conditions (e.g., those in blood), but are unstable below pH 5.5 or pH 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, eg, US Patent No. 5,622,929).

[0236] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935).

[0237] In other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0238] In other embodiments, the linker is non-cleavable and the effector molecule or detectable marker is released by degradation of the antibody (see U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety).

[0239] In several embodiments, the joint is resistant to cutting in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the joint is cut in the conjugate sample. Whether the joint is resistant to cutting in the extracellular environment can be determined, for example, as follows: the conjugate comprising the joint of interest is incubated with plasma for a predetermined time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours), and then the amount of free effector molecules or detectable markers present in the plasma is quantified. Different exemplary joints that can be used for conjugates are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0240] In several embodiments, conjugates of a CAR, a CAR-expressing T cell, an antibody, or an antigen-binding portion thereof, and one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, as well as derivatives of these toxins having toxin activity, are provided.

[0241] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to known methods, can be produced using genetic engineering techniques (see Yu et al., (2002) PNAS 99: 7968-7973), or can be maytansinol and maytansinol analogs prepared synthetically according to known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, each of which is incorporated herein by reference. Conjugates comprising maytansinoids, methods for their preparation, and their therapeutic uses are disclosed, for example, in US Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 Bl, the disclosures of which are expressly incorporated herein by reference.

[0242] Other toxins can be used together with CAR, the T cells expressing CAR, antibodies or their antigen binding portions.Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, nuclear toxins (ribotoxin), ribonuclease, saporin and calicheamicin, and botulinum toxin A to F. These toxins are well known in the art and many can be easily obtained from commercial sources (such as Sigma Chemical Company, St.Louis, MO). Expected toxins also include variants of toxins (see, for example, referring to U.S. Patent Nos. 5,079,163 and 4,689,401).

[0243] Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosome complex (Stirpe et al., Bio / Technology, 10: 405-412, 1992). However, the toxin does not have a specific mechanism for entering cells and therefore needs to be conjugated to an antibody or antigen-binding fragment that recognizes a cell surface protein to be internalized for efficient cell uptake.

[0244] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxins used in immunotoxins are mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.

[0245] Ricin is a lectin RCA60 from Ricinus communis (castor bean). For some examples of ricin, see U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, which are designated RCA based on their molecular weights of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to galactose residues on the cell surface and facilitates the transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0246] Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary nuclear toxins such as α-sarcin and restrictocin are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne family of antitumor antibiotics that cause double-strand breaks in DNA and lead to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0247] Abrin includes toxic lectins from the fruit of the plant Abrus precatorius. The toxic components, abrin a, b, c, and d, have molecular weights of approximately 63 kD to 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. Chain A inhibits protein synthesis; chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0248] CARs, T cells expressing CARs, monoclonal antibodies, and antigen-binding fragments thereof that are specific for one or more antigens disclosed herein can also be conjugated to a detectable marker; for example, a detectable marker that can be detected by: ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optics, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, available detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP) are also used. CAR, T cells expressing CAR, antibodies or their antigen-binding portions can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When CAR, T cells expressing CAR, antibodies or their antigen-binding portions are conjugated to a detectable enzyme, they can be detected by adding another reagent used by the enzyme to produce a distinguishable reaction product. For example, when there is reagent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. CAR, T cells expressing CAR, antibodies or their antigen-binding portions can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0249] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can be conjugated to paramagnetic agents (e.g., gadolinium). Paramagnetic agents (e.g., superparamagnetic iron oxide) are also used as labels. Antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope (e.g., a leucine zipper pair sequence, a binding site of a second antibody, a metal binding domain, an epitope tag) recognized by a second reporter.

[0250] CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can also be conjugated to radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect one or more antigens and antigen-expressing cells disclosed herein by x-rays, emission spectra, or other diagnostic techniques. In addition, radiolabels can be used therapeutically as toxins to treat tumors in subjects, for example, for the treatment of neuroblastoma. Some examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I.

[0251] Methods for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted illumination. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme acting on the substrate, and colorimetric labels are detected by simply visualizing the colored marker.

[0252] D. Nucleotides, expression, vectors and host cells

[0253] One embodiment of the present invention further provides a nucleic acid comprising a nucleotide sequence encoding any CAR described herein, antibody, or its antigen binding portion (including its functional portion and functional variant). The nucleic acid of the present invention may include a nucleotide sequence encoding any leader sequence, antigen binding domain, transmembrane domain and / or intracellular T cell signaling domain as described herein.

[0254] In some embodiments, the nucleotide sequence can be codon-modified. Without being bound by a particular theory, it is believed that the codon optimization of the nucleotide sequence improves the translation efficiency of the mRNA transcript. The codon optimization of the nucleotide sequence can involve replacing a natural codon with another codon, and the identical amino acid encoded by the other codon can be translated by the tRNA that is more easily obtained in the cell, thereby improving translation efficiency. The optimization of the nucleotide sequence can also reduce the secondary mRNA structure that may interfere with translation, thereby improving translation efficiency.

[0255] In one embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding the antigen binding domains of the CAR of the present invention. In another embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding any CAR described herein (including its functional part and functional variant).

[0256] As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a DNA or RNA polymer that can be single-stranded or double-stranded, synthetic, or obtained from a natural source (e.g., isolated and / or purified); it can contain natural, non-natural, or altered nucleotides; and it can contain natural, non-natural, or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions in some cases.

[0257] Recombinant nucleic acid can be the nucleic acid with non-natural sequence or with the sequence prepared by artificial combination of two otherwise separated sequence fragments.This artificial combination is usually by chemical synthesis or more commonly by artificial operation separation of nucleic acid fragments, for example, by genetic engineering technology, such as those described in Sambrook etc. (the same) realize.Nucleic acid can use operation known in the art to build based on chemical synthesis and / or enzymatic ligation.See, for example, Sambrook etc. (the same) and Ausubel etc. (the same).For example, nucleic acid can use naturally occurring nucleotides or be designed to improve the biological stability of molecule or improve the physical stability of the doublet formed after hybridization through different modified nucleotides (such as nucleotides substituted by thiophosphate derivatives and acridine). Some examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylquenoside, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, quenoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0258] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR or a functional portion or functional variant thereof. Alternatively, the nucleotide sequence may comprise a degenerate nucleotide sequence of any sequence or a combination of degenerate sequences.

[0259] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0260] The nucleotide sequence hybridized under stringent conditions can hybridize under high stringency conditions." high stringency conditions " means that the nucleotide sequence is specifically hybridized with the target sequence (nucleotide sequence of any nucleic acid described herein) with an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that can make the polynucleotides with accurate complementary sequences or the polynucleotides comprising only a few scattered mismatches be distinguished from the random sequence of the small regions (such as 3 to 10 bases) that occasionally match the nucleotide sequence. Compared with the full-length complement with 14 to 17 bases or more bases, such small complementary regions are more likely to melt, and high stringency hybridization makes it easily distinguishable. Relative high stringency conditions can include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalent at a temperature of about 50 DEG C to 70 DEG C. Such high stringency conditions allow for few (if any) mismatches between the nucleotide sequence and the template chain or the target chain, and are particularly suitable for detecting the expression of any CAR of the present invention. It is generally understood that conditions can be made more stringent by adding incremental formamide.

[0261] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

[0262] In one embodiment, nucleic acid can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any nucleic acid. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct comprising a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and allowing a host cell to express the mRNA, protein, polypeptide, or peptide when the construct comprises the nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and allows the host cell to contact the mRNA, protein, polypeptide, or peptide under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vector is not naturally occurring as a whole.

[0263] However, portions of the vector may be naturally occurring. Recombinant expression vectors may comprise any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which may comprise natural, non-natural, or altered nucleotides. Recombinant expression vectors may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0264] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell.Suitable carriers include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.Carrier can be selected from pUC series (Fermentas Life Sciences, GlenBurnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).

[0265] Phage vectors such as 1, λZapII (Stratagene), EMBL4 and λNMI 149. Some examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121 and pBIN19 (Clontech). Some examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector or a lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of a lentiviral genome, particularly including self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but not limited to, the LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica plc, the LENTIMAX.TM. vector system from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

[0266] A variety of transfection techniques are well known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al. (supra); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

[0267] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high-speed microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0268] In one embodiment, the recombinant expression vector can be prepared using standard recombinant DNA techniques such as those described in Sambrook et al. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0269] Recombinant expression vectors may contain regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0270] The recombinant expression vector may contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, such as antibiotic resistance, heavy metal resistance, and the like; complementation of prototrophy in auxotrophic hosts; and the like. Suitable marker genes for use in the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0271] The recombinant expression vector may comprise a natural or non-natural promoter operably connected to a nucleotide sequence encoding CAR (including its functional part and functional variant) or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding CAR. The selection of promoters (e.g., strong, weak, inducible, tissue-specific and development-specific) is within the ordinary skill of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the skill of the technician. Promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, or a promoter found in the long terminal repeat of mouse stem cell virus.

[0272] The recombinant expression vector can be designed for transient expression, for stable expression or for both. In addition, the recombinant expression vector can be made for constitutive expression or for inducible expression.

[0273] In addition, the recombinant expression vector can be prepared to contain a suicide gene. The term "suicide gene" as used herein refers to a gene that causes cell death in which the suicide gene is expressed. A suicide gene can be a gene that confers sensitivity to the cell expressing the gene, such as a drug, and causes cell death when the cell is contacted with or exposed to an agent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0274] One embodiment also provides a host cell comprising any recombinant expression vector described herein. The term "host cell" as used herein refers to any type of cell that can comprise the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus or algae, or can be a prokaryotic cell, such as a bacterium or a protozoan. The host cell can be a cultured cell or a primary cell, i.e., directly isolated from an organism (e.g., a human). The host cell can be an adherent cell or a suspension cell, i.e., a cell grown in suspension. Suitable host cells are known in the art and include, for example, DH5a Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5a cell. For the purpose of producing recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. While the host cell may be of any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0275] For the purposes of this document, T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. If obtained from a mammal, the T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. The T cells can be human T cells. The T cells can be T cells isolated from humans. The T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4 T cells. + / CD8 + Double-positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), naive T cells, etc. T cells can be CD8 + T cells or CD4 + T cells.

[0276] In one embodiment, the CAR described herein can be used for suitable non-T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells produced by pluripotent stem cells.

[0277] One embodiment also provides a cell mass comprising at least one host cell as described herein.Cell mass can be a heterogeneous colony, which comprises the host cell containing any recombinant expression vector and at least one other cell, such as a host cell (such as T cell) or a cell different from a T cell, such as a B cell, macrophage, neutrophil granulocyte, erythrocyte, hepatocyte, endothelial cell, epithelial cell, myocyte, brain cell, etc., not comprising any recombinant expression vector. Alternatively, cell mass can be a substantially homogeneous colony, wherein the colony mainly comprises the host cell (such as substantially consisting of) containing a recombinant expression vector. The colony can also be a clonal cell colony, wherein all cells in the colony are clones of the single host cell comprising a recombinant expression vector, so that all cells in the colony comprise a recombinant expression vector. In one embodiment of the invention, cell mass is a clonal colony comprising the host cell containing a recombinant expression vector as described herein.

[0278] CAR (including its functional part and variant), nucleic acid, recombinant expression vector, host cell (including its population) and antibody (including its antigen binding portion) can be separated and / or purified. For example, a purified (or separated) host cell preparation is a host cell preparation in which the host cell is purer than the cell in its natural environment in vivo. Such a host cell can be produced, for example, by standard purification techniques. In some embodiments, the preparation of the host cell is purified so that the host cell represents at least about 50% of the total cell content of the preparation, for example, at least about 70%. For example, the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.

[0279] E. Treatment methods

[0280] It is expected that the CAR disclosed herein can be used in a method for treating or preventing a disease in a mammal. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or antigen binding portion thereof, and / or pharmaceutical composition to the mammal in an amount effective to treat or prevent cancer in the mammal.

[0281] One embodiment also includes subjecting the mammal to lymphodepletion prior to administering the CAR disclosed herein. Some examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepletion chemotherapy, myeloablative lymphodepletion chemotherapy, whole body irradiation, and the like.

[0282] For the purpose of the method for wherein administering host cell or cell mass, cell can be mammalian allogeneic or autologous cell.Preferably, cell is mammalian autologous.As used herein, allogeneic means following any material, and it and the individual that this material is introduced are derived from different animals of same species.When the gene of one or more loci is not identical, two or more individuals are considered to be allogeneic to each other.In some respects, the allogeneic material from the individual of same species can be fully dissimilar in genetics to interact on antigenicity.As used herein, " autologous " means following any material, and it derives from the same individual that this material is reintroduced into later.

[0283] The mammals referred to herein can be any mammal. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters; and mammals of the order Lagomorpha, such as rabbits. The mammal can be from the order Carnivora, including Felidae (cats) and Canidae (dogs). The mammal can be from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs); or from the order Persistodactyla, including Equine (horses). The mammal can be from the order Primates, Ceboids, or Simoids (monkeys); or from the order Anthropoids (humans and apes). Preferably, the mammal is a human.

[0284] With respect to the methods, the cancer can be any cancer, including any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's Lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0285] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention recognized by one of ordinary skill in the art as potentially beneficial or therapeutic. In this regard, the methods can provide any amount or level of cancer treatment or prevention in a mammal.

[0286] In addition, the treatment or prevention provided by the method can include treating or preventing one or more conditions or symptoms of the disease (e.g., cancer) being treated or prevented. In addition, for purposes herein, "prevention" can encompass delaying the onset of a disease or its symptoms or conditions.

[0287] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; (b) and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0288] Samples can be obtained by any suitable method (e.g., biopsy or autopsy). A biopsy is the removal of tissue and / or cells from an individual. Such removal can be for the purpose of collecting tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells. The experiments can include experiments to determine whether an individual has and / or is suffering from a particular condition or disease state. The condition or disease can be, for example, cancer.

[0289] For one embodiment of a method for detecting the presence of a proliferative disorder (e.g., cancer) in a mammal, the sample comprising cells of the mammal can be a sample comprising whole cells, a whole cell lysate, or a fraction of a whole cell lysate (e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction). If the sample comprises whole cells, the cells can be any cell of the mammal, e.g., a cell of any organ or tissue, including a blood cell or an endothelial cell.

[0290] For mammals, contacting can occur in vitro or in vivo. Preferably, contacting is in vitro.

[0291] In addition, the detection of the complex can be carried out by any number of ways known in the art. For example, CAR disclosed herein, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell mass or antibody or its antigen binding portion thereof as described herein can be marked with a detectable marker, such as a radioisotope, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and element particles (e.g., gold particles) as disclosed above.

[0292] The method for testing the ability and antigen specificity of CAR to recognize target cells is known in the art. For example, Clay et al., J.Immunol, 163: 507-513 (1999) teach the method for measuring the release of cytokines (such as interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)). In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J.Immunol.174: 4415-4423 (2005).

[0293] Another embodiment provides the CAR of the present invention, nucleic acid, recombinant expression vector, host cell, cell group, antibody or its antigen binding portion thereof, and / or pharmaceutical composition for treating or preventing proliferative disorders (eg, cancer) in mammals. Cancer can be any cancer described herein.

[0294] Any method of administration can be used for the disclosed therapeutic agent, including local and systemic administration. For example, surface, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used. The specific mode of administration and dosage regimen will be selected by the attending clinician considering the specific circumstances of the case (e.g., object, disease, involved disease state and whether the treatment is preventive). In the case where more than one agent or composition is administered, one or more routes of administration can be used; for example, chemotherapeutic agents can be administered orally and antibodies or antigen binding fragments or conjugates or compositions can be administered intravenously. Methods of administration include injection, in which case CAR, CAR T cells, conjugates, antibodies, antigen binding fragments or compositions are provided in, for example, the following non-toxic pharmaceutically acceptable carriers: water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate or liposomes. In some embodiments, local administration of the disclosed compound can be used, for example, by applying the antibody or antigen binding fragment to a tissue area from which the tumor has been removed, or to an area suspected of being prone to tumorigenesis. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical formulation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops, or intravitreally to the eye.

[0295] The disclosed therapeutic agents can be formulated into unit dosage forms suitable for single administration of precise dosages. In addition, the disclosed therapeutic agents can be administered in a single dose or in a multiple dose regimen. A multiple dose regimen is a regimen in which the primary course of treatment may have more than one single dose, for example 1 to 10 doses, followed by additional doses administered at subsequent intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound over a period of days to months or even years. Therefore, the dosage regimen will also be determined, at least in part, based on the specific needs of the subject to be treated and will depend on the judgment of the administering physician.

[0296] A typical dosage of the antibody or conjugate may be from about 0.01 mg / kg to about 30 mg / kg, for example from about 0.1 mg / kg to about 10 mg / kg.

[0297] In some specific examples, a therapeutic composition comprising one or more conjugates, antibodies, compositions, CARs, CAR T cells, or other agents is administered to a subject for a period of, for example, weeks, months, or years, based on a multiple daily dosing regimen (e.g., at least 2 consecutive days, 10 consecutive days, etc.). In one example, a conjugate, antibody, composition, or other agent is administered to a subject for at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0298] In some embodiments, the disclosed method includes providing surgery, radiotherapy and / or chemotherapy to the object in combination with disclosed antibodies, antigen binding fragments, conjugates, CAR or T cells expressing CAR (e.g., sequentially, substantially simultaneously or simultaneously). Such medicaments and methods for treatment and therapeutic doses are well known to those skilled in the art and can be determined by skilled clinicians. The preparation and dosage regimen for other medicaments can be used according to the manufacturer's instructions or as determined empirically by technicians. Preparations and dosage regimens for such chemotherapy are also described in Chemotherapy Service, (1992) editor, MCPerry, Williams & Wilkins, Baltimore, Md.

[0299] In some embodiments, the combination therapy may include administering a therapeutically effective amount of another cancer inhibitor to the subject. Non-limiting examples of other therapeutic agents that may be used for combination therapy include microtubule binding agents, DNA intercalators or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered in a therapeutically effective amount) and treatments may be used alone or in combination. For example, any suitable anticancer agent or anti-angiogenic agent may be administered in combination with CAR, CAR-T cells, antibodies, antigen binding fragments, or conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and may be determined by skilled clinicians.

[0300] Additional chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, dichloromethane, procarbazine, temozolomide, thiotepa, and uracil mustard; antimetabolites such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., clopidogrel, chloramphenicol, and chloramphenicol); clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and oxaliplatin); anthraquinone and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and other agents, such as alitretinoin, hexamethylmelamine, amsacrine, anagrelide, trimethoprim, and tretinoin. Arsenic oxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukindiftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masopropylamine, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib and tretinoin. The selection and therapeutic dosage of such agents are known to those skilled in the art and can be determined by a skilled clinician.

[0301] Combination therapy can provide a synergistic effect and demonstrate synergy, i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. Synergy can be achieved when the active ingredients are: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered in alternating or parallel delivery as separate formulations; or (3) by some other regimen. When delivered in alternation, synergy can be achieved when the compounds are administered or delivered sequentially, e.g., sequentially in separate syringes by different injections. Generally, during alternation, an effective dose of each active ingredient is administered sequentially, i.e., one after the other, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0302] In one embodiment, an effective amount of an antibody or antigen-binding fragment or its conjugate that specifically binds to one or more antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed to allow the administered antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on the corresponding cancer cells, immune complexes are detected. The presence (or absence) of immune complexes indicates the effectiveness of treatment. For example, an increase in immune complexes compared to a control obtained before treatment indicates that the treatment is ineffective, whereas a decrease in immune complexes compared to a control obtained before treatment indicates that the treatment is effective.

[0303] F. Biopharmaceutical Compositions

[0304] Provided herein are biopharmaceutical compositions or biopharmaceutical compositions (hereinafter "compositions") for gene therapy, immunotherapy and / or cell therapy, comprising one or more disclosed CARs specifically bound to one or more antigens disclosed herein, or T cells expressing CAR, antibodies, antigen binding fragments, conjugates, CARs, or T cells expressing CAR in a carrier (e.g., a pharmaceutically acceptable carrier). The composition can be prepared in a unit dose form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired result. The composition can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or T cells expressing CARs, antibodies, antigen binding fragments, conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising CARs disclosed herein, or T cells expressing CARs, conjugates, antibodies, or antigen binding fragments are used for, for example, the treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some instances, the composition can be used for the treatment or detection of cancer. Compositions comprising a CAR, or a T cell expressing a CAR, conjugate, antibody, or antigen-binding fragment as disclosed herein are also useful, for example, in the detection of pathological angiogenesis.

[0305] The composition for administration may include a solution of CAR or T cells expressing CAR, conjugates, antibodies or antigen binding fragments dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions may be sterilized by conventional known sterilization techniques. These compositions may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of CAR or T cells expressing CAR, antibodies or antigen binding fragments or conjugates in these preparations may vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the specific mode of administration selected and the needs of the subject. The actual method for preparing such a dosage form for gene therapy, immunotherapy and / or cell therapy is known or will be obvious to those skilled in the art.

[0306] Typical compositions for intravenous administration include about 0.01 mg / kg to about 30 mg / kg of antibody or antigen-binding fragment or conjugate / subject / day (or corresponding doses of CAR comprising the antibody or antigen-binding fragment, or T cells expressing CAR, conjugate). Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, PA (1995).

[0307] CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates can be provided in lyophilized form and rehydrated with sterile water before administration, but they are also provided as sterile solutions of known concentrations. The CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugate solutions are then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 mg / kg to 15 mg / kg body weight. There is considerable experience in the art with regard to the administration of antibodies or antigen-binding fragments and conjugate drugs; for example, since 1997, Since its approval, antibody drugs have been sold in the United States. CAR, or T cells expressing CAR, antibodies, antigen binding fragments and conjugates thereof can be administered by slow infusion rather than by intravenous push or bolus injection. In one example, a higher loading dose is administered, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg antibody or antigen binding fragment (or a corresponding dose of a conjugate comprising an antibody or antigen binding fragment) can be infused over a period of about 90 minutes, followed by a weekly maintenance dose of 2 mg / kg infused over 30 minutes if the previous dose is well tolerated for 4 to 8 weeks.

[0308] Controlled release parenteral formulations can be prepared as implants, oily injections or as particle systems. For an overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain a therapeutic protein (e.g., a cytotoxin or a drug) as a central core. In the microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively. The capillary diameter is about 5 μm so that only nanoparticles are administered intravenously. The diameter of the microparticles is generally about 100 μm and is administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339 (1992).

[0309] Polymers can be used for ion controlled release of CAR disclosed herein or T cells expressing CAR, antibodies or antigen binding fragments or conjugate compositions. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous but movable liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown that it is an effective carrier for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44 (2): 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). On the other hand, liposomes are used for the controlled release of lipid-encapsulated drugs as well as drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).

[0310] G.Medicine box

[0311] In one aspect, a kit for using the CAR disclosed herein is also provided. For example, the kit is used to treat a tumor in a subject, or to prepare a CAR T cell expressing one or more CARs disclosed herein. As disclosed herein, the kit will generally contain the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. The kit may contain more than one disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing CAR.

[0312] The medicine box may include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials (e.g., glass or plastic). The container typically holds a composition comprising one or more disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. In some embodiments, the container may have a sterile entrance (e.g., a container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0313] The label or package insert will generally also include instructions for use of disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CAR or T cells expressing CAR in a method for treating or preventing tumors or preparing CAR T cells. The package insert is generally included in instructions generally included in the commercial packaging of the therapeutic product, which includes information about the indications, usage, dosage, administration, contraindications and / or warnings related to the use of such therapeutic products. The illustrative material can be written in electronic form (such as a computer disk or a compact disc) or can be visual (such as a video file). The medicine box can also include other components that are conducive to the specific application for which the medicine box is designed. Therefore, for example, the medicine box can additionally include tools for detecting the label (such as enzyme substrates for enzyme labeling, filter devices for detecting fluorescent labels, suitable second labels (such as second antibodies), etc.). The medicine box can additionally include buffers and other reagents commonly used in the practice of ad hoc methods. Such medicine boxes and suitable contents are well known to those skilled in the art.

[0314] Example

[0315] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention in any way. On the contrary, it should be clearly understood that a number of other embodiments, modifications thereof, and equivalents thereof may be sought, which may be apparent to those skilled in the art after reading the description herein without departing from the spirit of the present invention and / or the scope of the appended claims.

[0316] Example 1. Isolation of CD33-specific antibodies from fully human phage-displayed ScFv and VH libraries

[0317] Materials and methods:

[0318] a) Generation of human phage-displayed ScFv and VH CD33-specific antibodies

[0319] A naive human ScFv (recombinant single-chain variable fragment of immunoglobulin) phage display library (approximately 10 diversity) constructed from peripheral blood B cells from 50 healthy donors was used. 10 ScFv or VH specific for recombinant human CD33 was selected by combining 10 12During the first, second, and third rounds of biopanning, amplified libraries of ScFv or VH displayed by each phage were incubated with 5, 3, and 1 μg of coated CD33 in a 5×100 μl volume evenly distributed in 5 wells of a 96-well plate at room temperature for two hours. After each round of incubation, the wells were washed 5 times with phosphate-buffered saline containing 0.05% Tween 20 (PBST) for the first round and 10 times for the subsequent rounds to remove nonspecifically bound phage. The bound phage was mixed with TG1 competent cells at 37°C for 1 hour, phage was amplified from the infected cells, and used for the next round of biopanning. After the third round of biopanning, 380 clones were randomly picked from infected TG1 cells using the automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI) and each inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate. After the bacterial culture reached an optical density of 0.5 at 600 nm (OD600), helper phage M13K07 at a multiplicity of infection (MOI) of 10 and 50 μg / ml (final concentration) of kanamycin were added to the culture medium, and the plates were further incubated overnight at 30°C on a shaker at 250 rpm. The phage supernatant was mixed with 3% skim milk in PBS at a volume ratio of 4:1 and used in enzyme-linked immunosorbent assay (ELISA) to identify phage clones displaying ScFv or VH with high CD33 binding affinity. The supernatant was incubated with 50 ng of coated recombinant human CD33 per well in a 96-well plate at room temperature for 2 hours and washed five times with PBST (after overnight incubation at 4°C, the plates were blocked with 3% skim milk in PBS and washed three times with PBS containing 0.05% Tween 20). CD33-bound phage were detected using a horseradish peroxidase-conjugated goat anti-M13 antibody. After incubation with the antibody, nonspecifically bound antibody was removed by washing the wells, and 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added, and the absorbance of the solution at 450 nm (A450) was measured. Clones that bound to CD33 with an A450 > 1.0 were selected for further characterization.

[0320] b) Expression and purification of selected soluble ScFv or VH

[0321] DNA sequencing was performed on the VH and VL of the selected clones, as well as the VH of the domain binders, and the ScFv or VH with unique sequences encoded by the clones was expressed and purified as described below. Plasmids extracted from these clones were used to transform HB2151 cells. A single colony was picked from a plate containing freshly transformed cells and inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose and incubated at 37°C with shaking at 250 rpm. When the culture reached an OD of 0.90 at 600 nm, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was further incubated at 30°C overnight. The bacterial pellet was collected after centrifugation at 8,000 × g for 20 minutes and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation at room temperature for 30 minutes with rotation at 50 rpm, the resuspended pellet was centrifuged at 25,000 xg for 25 minutes at 4°C, and the supernatant was used for ScFv purification using Ni-NTA resin according to the supplier's protocol (Qiagen).

[0322] c) ELISA binding assay

[0323] Diluted recombinant human CD33 in 50 μl of PBS at 2 μg / ml was coated overnight in a 96-well plate at 4°C. Purified ScFv or VH with His and Flag tags (from above) were serially diluted and added to the target protein-coated wells. After washing, a 1:3000 dilution of HRP-conjugated anti-Flag antibody was added for 1 hour at room temperature. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes. 1N H2SO4 was added to stop the reaction, and the OD was read at 450 nm to quantify the relative ability of the ScFv to bind to CD33.

[0324] result:

[0325] Based on the results of ELISA binding assays, four individual ScFs clones specific for recombinant human CD33 were identified and labeled as human anti-CD33 ScFv binders m1033-9 (ScFv9), m1033-10 (ScFv10), m1033-12 (ScFv12), and m1033-15 (ScFv15), respectively. Two unique VH domain binders, m1033-2 (VH-2) and m1033-4 (VH-4), were also identified from the ELISA binding assays. The generation of chimeric antigen receptors expressing VH-2, VH-4, ScFv9, ScFv10, ScFv12, and ScFv15 human anti-CD33 binders is outlined in Example 2 below.

[0326] Example 2. Expression of anti-CD33 CARs based on fully human heavy chain-only Ig or scFv-based binding sequences

[0327] In this example, a novel fully human immunoglobulin heavy chain-only anti-CD33 CAR T cell derived from a single-chain variable fragment (scFv) binder sequence is described. The novel anti-CD33 CART construct has demonstrated high-level expression in primary human T cells and has specificity and potent cytotoxic and cytokine functions against CD33-positive tumor cells.

[0328] Homo sapiens CD33 (sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67) is a well-studied target in acute myeloid leukemia (AML). Humanized CD33 antibodies (lintuzumab) and CD33 antibody-drug conjugates (gemtuzumab or GO, Pfizer) have shown some efficacy but have failed to demonstrate robust therapeutic benefit in clinical trials (1. Feldman EJ et al., J Clin Oncol 2005; 23(18): 4110-4116, 2. Petersdorf SH et al., Blood 2013; 121(24): 4854-4860). AMG330, a CD33-CD3 bispecific T cell engager (BiTE), is also under investigation (Krupka C et al., Blood 2014123: 356-365). As of this year, GO has been reintroduced into the clinic with an altered, much lower dose and modified regimen, but sufficient clinical data are needed to re-evaluate this agent. Another agent currently in development, the CD33-targeting antibody-drug conjugate vadastuximab talirine (SGN-CD33A), was recently placed on clinical hold in some Phase I / II clinical trials due to hepatotoxicity (available on the World Wide Web at investor.seattlegenetics.com / phoenix.zhtml?c=124860&p=irol-newsArticle&ID=2232880), highlighting the urgent need to identify safe and effective CD33-targeting modalities.

[0329] CD33 CARs were designed using CD33-binding sequences derived from immunoglobulin VH domains or full-length ScFv under the control of the EF1a promoter and tested in vitro for transduction efficiency, killing function, and cytokine production.

[0330] Materials and methods:

[0331] (a) Cell lines

[0332] The human cell line promyelocytic leukemia HL-60, acute lymphoblastic leukemia Reh, monocytic leukemia THP-1 and myeloid leukemia K562 cell line are purchased from American Tissue Culture Collection (American Tissue Culture Collection) (ATCC, Manassas, VA). Acute myeloid leukemia MOLM-14 is purchased from German microorganism and cell line collection (German Collection of Microorganisms and Cell Lines) (DSMZ, Braunschweig Germany). Cultured cell line in the RPMI-1640 culture medium (ATCC) supplemented with 10% heat-inactivated fetal bovine serum. The THP-1 culture medium also comprises 0.05% beta-mercaptoethanol. By using a lentiviral vector (Lentigen Technology, Inc., Gaithersburg, MD) stably transduced wild-type leukemia cell line with or without the encoding firefly luciferase of GFP, limiting dilution and selection of luciferase positive clones produce luciferase expression subclones subsequently.

[0333] (b) Construction of chimeric antigen receptor (CAR)-expression vector

[0334] CAR antigen binding domain sequence is derived from human anti-CD33 ScFv or heavy chain variable fragment.By connecting the binding substance sequence with CD8a and membrane spaning domain (UniProt sequence ID P01732, aa 138-206), then with 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signal transduction domain and CD3 ζ signal transduction domain (CD247, aa52-163, reference sequence ID: NP_000725.1) frame connection to produce CAR T construct.For some constructs, CD28 costimulatory sequences are used instead of 4-1BB costimulatory sequences.In some constructs, CD8 is connected and / or membrane spaning domain is replaced with the domain derived from TNFRSF19 protein. For some sequences, the truncated epidermal growth factor receptor (tEGFR) tag was incorporated into the CAR construct via a 2A peptide to enable in vitro labeling of transduced cells and as a suicide switch for in vivo applications. The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing lentiviral vectors (LVs) were generated by transient transfection of HEK 293T cells, and the supernatants containing lentiviral vectors were precipitated by centrifugation and stored at -80 °C.

[0335] (c) Purification and transduction of primary T cells

[0336] Human primary T cells from normal donors were cultured for CD4 + and CD8 + Cells were purified from buffy coats after immunomagnetic selection and cultured at 0.3 × 10 6 cells / ml to 2x10 6 The cells were cultured at a density of 10 cells / ml and CD3 / CD28 GMP TransAct reagent (Miltenyi Biotec) was used for activation and transduction with a lentiviral vector encoding a CAR construct overnight in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO) on day 2, with a medium change on day 4. On day 3, the cultures were transferred to TexMACS medium supplemented with 200 IU / ml IL-2 and propagated until harvest on days 7 to 10.

[0337] (d) Immune effector assays (CTL and cytokines)

[0338] To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells in a variety of effector / target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence was quantified as counts per second (sample CPS). Target-only wells (maximum CPS) and target-only wells plus 1% Tween-20 (minimum CPS) were used to determine the assay range. Percent lysis was calculated as (1-(sample CPS-minimum CPS) / (maximum CPS-minimum CPS)). The supernatant of the co-culture with an E:T ratio of 10:1 was removed and the concentrations of IFNγ, TNFα, and IL-2 were analyzed by ELISA (eBioscience, San Diego, CA).

[0339] (e) Flow cytometric analysis of CAR surface expression

[0340] For cell staining, half a million CAR T transduced cells were harvested from culture, washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and CAR surface expression was detected by staining with CD33-Fc peptide (R&D, Minneapolis, MN) and then with anti-Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). Untransduced cells were used as negative controls. Dead cells were excluded from all studies by 7AAD staining (BD Biosciences, San Jose, CA). The cells were washed twice and resuspended in 200 μl of staining buffer and then quantitatively analyzed by flow cytometry. Flow cytometric analysis was performed on a 10 analyzer (Miltenyi Biotec), and data graphs were generated using FlowJo software (Ashland, OR).

[0341] (f) In vivo analysis of CAR T function

[0342] The function of CD33-targeted CAR T cells was evaluated in vivo. 6- to 8-week-old NSG mice (6 per group) were inoculated with 1.0 × 10 6 MOLM-14 CD33 + On day 4, tumor burden was determined by IVIS bioluminescence imaging, and mice were randomized into groups with equal mean tumor burden and 5.0 x 10 6 CAR T +Cells / mouse. On days 14, 21, 28, and 35, tumor regression was determined by bioluminescent imaging. The survival of the mice was recorded and analyzed at the end of the study. To determine the presence of CAR T and tumor cells, blood was collected from all animals on day 19 of the study. The absolute number of blood CAR T cells and MOLM-14 tumor cells was determined by flow cytometry, and the levels of inflammatory cytokines were measured in plasma by MACSPlex cytokine 12 human kit (Miltenyi Biotec) according to the manufacturer's protocol.

[0343] (g) Flow cytometric analysis of CAR T and tumor cells in mouse blood

[0344] For flow cytometry, 50 μl of blood was collected and analyzed for CAR T and MOLM-14 tumor cell numbers. First, erythrocytes were lysed with erythrocyte lysis buffer (Miltenyi Biotec) according to the manufacturer's instructions, and human CD45 + , CD3 + The leukocyte of 100 cells / wells is stained with 7-AAD (Miltenyi Biotec) and BD Biosciences, San Jose, CA).The leukocyte of 100 cells / wells is stained with 7-AAD (Miltenyi Biotec), and is obtained by MACSQiant 10 flow cytometers (Miltenyi Biotec).The MOLM-14-cell of stably expressing GFP reporter gene is detected in the B1 passage.Seven AAD positive dead cells have been excluded from analysis.In order to promote the direct quantification of human T cell and MOLM-14 number in blood, before obtaining, CountBright absolute counting beads (ThermoFischer Scientific, Waltham, MA) are added to each sample, and the corresponding absolute cell number is calculated according to the scheme of manufacturers.

[0345] (h) Long-term CAR T and tumor co-incubation assay

[0346] CART cell lines expressing various anti-CD33 CAR constructs and controls were combined with tumor target HL-60 cells at an effector to target ratio of 5:1 to 0.04:1 for 5 or 11 days. Negative control UTD (untransduced cells), T cells alone (E:T 1:0) and T cells expressing GFP (1398) were included. At each time point, the cells were stained with anti-human CD33 and CD3 antibodies and 7-AAD and acquired on a MACSQuant 10 flow cytometer. In order to determine the percentage of surviving CAR T cells and tumor cells in each case, the cells were stained in the forward and side scatter single peak 7-AAD. - 、CD3 + or CD33 + Set up the gate.

[0347] result:

[0348] In order to evaluate the new anti-CD33 fully human ScFv binding sequence, each of the heavy chain binding sequence VH-2 or VH-4 or ScFv sequence ScFv9, ScFv10, ScFv12 or ScFv15 was designed as a CAR construct for tumor antigen binding domains. In each CAR design, the tumor targeting domain is followed by a joint and a transmembrane domain, a 4-1BB costimulatory domain and a CD3 ζ signal transduction domain (Table 1 below) derived from human CD8 protein. Construct LTG1940 incorporating the ScFv binding domain derived from sequence My96 was used as a reference control or comparator.

[0349] Table 1: List of CD33-targeted CAR constructs

[0350] LTG1905: EF1a VH-2 CD33-CD8 TM-41BB-CD3ζ LTG1906: EF1a VH-4 CD33-CD8TM-4-1BB-CD3ζ LTG1936: EF1a-ScFv9-CD8TM-4-1BB-CD3ζ LTG1937: EF1a-ScFv10-CD8TM-4-1BB-CD3ζ LTG1938: EF1a-ScFv12-CD33 CAR-CD8TM-4-1BB-CD3ζ LTG1939: EF1a-ScFv15-CD33 CAR-CD8TM-4-1BB-CD3ζ LTG1940: EF1a-My96_ScFv-CD33 CAR-CD8TM-4-1BB-CD3ζ

[0351] T cells transduced with anti-CD33 chimeric antigen receptor exhibited surface expression and cytolytic activity.

[0352] a) Surface expression of anti-CD33 CAR

[0353] To evaluate the new anti-CD33 CAR, a lentiviral vector (LV) encoding a CAR construct under the control of the human EF1a promoter was generated as described in the Materials and Methods. Then, human primary T cells from two independent healthy donors were transduced with four lentiviral vectors encoding CAR. Non-transduced cells (NT) from the same donor or GFP-transduced cells from the same donor were used as negative controls.

[0354] As described in the Materials and Methods, T cells were activated with TransAct T cell reagent (an active linker of CD3 and CD28 antigens, Miltenyi Biotec, Inc.) on culture day 0 in the presence of IL-2. On culture day 10, expression of anti-CD33 CAR on the T cell surface was detected by CD33-Fc peptide followed by anti-Fc-AF647 and analyzed by flow cytometry. Anti-CD33 CAR constructs exhibited surface CAR expression.

[0355] b) Cell lysis assay of anti-CD33 CAR

[0356] To demonstrate the cytolytic function of the generated CAR T cells, a luciferase-based killing assay was performed using HL-60-luc, MOLM-14 (CD33-high), Reh-luc, and K562-luc (CD33-low) leukemia cell lines stably expressing firefly luciferase. CART cells were combined with target cells at effector to target (E:T) ratios of 20, 10, and 5 and incubated overnight, and cell killing was assessed by luminescence as described in Materials and Methods ( Figure 3 Figure 4 ; Figure 6 and Figure 7 ). When testing VH-based anti-CD33 CARs, the CAR T construct LTG1906 exhibited strong E:T ratio-dependent cytotoxicity against the high CD33 HL-60-luc cell line, moderate cell lysis against the lower CD33-expressing K562 cell line, and only weak cell lysis activity against the low CD33 Reh-luc cell line. Therefore, cell lysis activity is directly correlated with the CD33 expression level of the leukemia. In addition, the negative control GFP constructs LTG1398 and NT (untransduced T cells from the same donor) had no cell lysis effect, indicating that the cytotoxicity was CART-dependent. Notably, the LTG1905 CAR construct was not cell lytic in the HL-60 luc cell line and had only weak cell lysis in the K562-luc cell line.

[0357] Likewise, construct LTG1906 produced high levels of IFNγ, TNFα, and IL-2 in response to CD33-positive tumor lines THP-1 and HL-60, whereas CAR T-secreting cytokines remained at low levels when challenged with leukemia cell lines K562 or Reh expressing low levels of CD33 antigen ( Figure 5 Interestingly, construct LTG1905, although inefficient in killing CD33-positive HL-60 leukemias in vitro, produced very high levels of IFNγ, TNFα, and IL-2 as detected by ELISA. Thus, the design of the CAR and the choice of binder are not trivial, as some binders are active in soluble IgG or ScFv form and can be expressed on the surface of T cells in CAR T format, but they are inefficient in killing CD33-positive tumors.

[0358] In contrast, when ScFv anti-CD33 CAR T cells were tested, constructs LTG1936 and LTG1939 exhibited robust killing activity against the high CD33 tumor cell lines HL-60 and MOLM-14, but much lower activity against the low CD33 Reh tumor cell line and barely detectable activity against the low CD33 K562 cells ( Figure 7). Surprisingly and unexpectedly, CAR constructs LTG 1937 and LTG 1938 were inefficient in lysing CD33-positive tumor targets. This again demonstrates that the design of CAR T constructs based on antibody fragments is not trivial, as soluble antibody binding properties and / or solubility and / or multimerization properties may not directly translate into CAR function. Similar to the VH-only construct 1906, the scFv-based constructs 1936, 1939 and the My96 scFv-based comparator construct 1940 were ineffective in lysing CD33-positive tumor targets. + When challenged, the tumor cell lines HL-60 and MOLM14 both produced high levels of IFNγ and TNFα, but the CD33 低 In the presence of cell line Reh or when CART cells are hatched alone in the absence of target cells, there is almost no cytokine induction. The CAR constructs 1937, 1938 of poor in vitro killing function have been shown to produce cytokines in response to tumor cells in an inefficient manner (data not shown). Compared with the cytokine induction of MOLM14 and HL60, MOLM14 shows a higher CD33 antigen density (30,000 sites per cell in MOLM14 compared to 25,000 sites per cell in HL-60, data not shown), which corresponds to the larger IFNγ and TNFα induction caused by MOLM14 for all anti-CD33 constructs tested. Similarly, this demonstrates the antigen-specific nature of anti-CD33 CAR activation. Unexpectedly, the induction of IL-2 is strong for CAR constructs 1906 and 1940, but is medium for CAR constructs 1936 and 1939.

[0359] Then, CAR T cells incorporating different constructs were cultured with HL-60 CD33 + Tumor cells were combined at E:T ratios ranging from 5:1 to 0.04:1 for long-term co-incubation assays. Untransduced T cells UTD, GFP-transduced T cells 1398, and T cells alone at E:t 0.1 were used as assay controls. Cells were co-cultured for 5 days (data not shown) or 11 days, and for each CAR construct, both days showed a similar trend of HL-60 elimination ( Figure 9). In the negative control groups (UTD and 1398), tumor cells grew rapidly and T cells disappeared, indicating that CAR-mediated T cell stimulation is required for cell lytic activity and prolonged CAR T survival. CAR constructs 1398, 1398, which performed poorly in the overnight in vitro assay, were ineffective in HL-60 killing in this long-term assay, and similar to the performance of the negative control group, CAR T cells disappeared from the culture and tumors persisted at all E:T ratios equal to or below 1:1. In contrast, anti-CD33 CAR constructs 1906, 1936, and 1939 were equally effective as comparator construct 1940 in CTL function and successfully eliminated HL-60 tumor cells at E:T ratios as low as 0.2:1 ( Figure 9 ). Therefore, constructs 1906, 1936, 1939, and 1940 were selected for further evaluation in an in vivo model of AML.

[0360] To facilitate the comparison of anti-CD33-CAR constructs in vivo, a xenograft mouse model was used as described in Materials and Methods. Briefly, NSG mice were inoculated with MOLM-14 cells stably expressing firefly luciferase and GFP on day 0, and 5 million CAR T cells were administered per mouse on day 5 of the study. Tumor growth kinetics were measured by IVIS bioluminescence imaging on days 14, 21, 28, and 35 of the study, and CAR T function was assessed in the blood of mice on day 19 of the study.

[0361] like Figure 10 As shown in Figure A, mice transplanted with MOLM-14 tumors and untreated (TA) or administered untransduced T cell controls (UTD) died of disease on day 14 of the study. CAR constructs 1936 and 1939 showed partial efficacy and delayed tumor growth and prolonged survival. Notably, CAR construct 1906 and comparator construct 1940 mediated MOML-14 tumor rejection, and all animals in these groups survived to the end of the study on day 39 ( Figure 10 A and B).

[0362] Blood was collected from each animal on day 19 of the study to assess the levels of CAR T cells in the blood, the levels of MOLM-14 tumor cells in the blood, and the levels of blood cytokines secreted by CAR T in each treatment group. The absolute number of CAR T cells and tumor cells in the blood samples was measured by flow cytometry ( Figure 11Although the number of CAR T cells in each group did not differ significantly, construct 1906 expressing CAR T cells tended to be higher than the other groups, followed by the CAR comparator construct 1940. Interestingly, T cell levels were also higher in the UTD control group, which contained untransduced T cells, likely due to the large number of cells initially infused in this group (8.0 x 10 6 Notably, we detected a statistically significant reduction in the number of circulating MOLM-14 tumor cells in all CAR T groups compared with the UTD control ( Figure 11 (A, right panel). Furthermore, when the CAR T groups were compared to each other, CAR 1906 and 1940 stimulated the strongest reduction of MOLM-14 cells, which was significantly greater than CAR 1936 and 1939. Thus, CAR 1906 and 1940 were the most effective in controlling blood MOLM-14 levels, followed by CAR 1936 and CAR 1939.

[0363] Measurable levels of the inflammatory cytokines GM-CSF, IFNγ, and IL-2 were detected in mice dosed with CAR T cells or UTD control. Although the differences between the levels of these cytokines were not significant, plasma GM-CSF and IFNγ levels tended to be higher with construct 1906, while IL-2 levels tended to be elevated with CAR1906 and CAR constructs 1940 and 1936 ( Figure 11 These results highlight the enhanced secretion of inflammatory cytokines by activated CAR T cells. No significant differences were detected between the experimental groups, possibly because CAR T cells may have exceeded maximal activation by study day 19 (it is worth noting that differences in tumor burden were detected as early as study day 14, Figure 10 A), however, the most effective CARs 1906 and 1940 in tumor rejection also tended to secrete higher levels of cytokines.

[0364] In summary, the high functionality of the new fully human anti-CD33 CAR construct LTG1906, as well as the partial functionality of constructs LTG1936 and LTG1939, was confirmed in vitro and in vivo (Table 2 below). It is conceivable that the functionality of constructs LTG1936 and LTG1939 can be further improved by redesigning the CAR spacer, linker, or co-stimulatory domain to allow better access to the specific epitope targeted thereto or to increase the level of CAR response to tumor epitope binding. Despite having detectable surface expression and high cytokine secretion by flow cytometry, the VH-based anti-CD33 CAR construct LTG1905 has a low cell lysis effect. Despite high expression, the ScFv-based CAR T constructs LTG1937 and LTG1938 are also inefficient in lysing target cell lines in vitro.

[0365] Example 3. Improved functional properties of the CD33 CAR portion can be achieved by altering the structure of the anti-CD33 CAR to express a fully human heavy chain-only or ScFv-based binding sequence

[0366] In this example, different structural configurations of anti-CD33 CAR T cells derived from novel fully human immunoglobulin heavy chain-only or scFv binder sequences are described.

[0367] When challenged with tumor cells expressing antigens, for example, by incorporating a single CD28-derived vs CD137 / 4-1BB-derived costimulatory domain (second generation CAR) in frame with an activation domain (e.g., CD3ζ), a CAR structure lacking a costimulatory domain (first generation CAR), or incorporating multiple tandem costimulatory domains (third generation CAR), it is hypothesized that CART cells secrete higher or lower levels of inflammatory cytokines, such as IL-2, IFNγ, TNFα.

[0368] In some constructs, the incorporation of novel hinge and transmembrane domains (e.g., domains derived from the human TNFRSF19 sequence) can confer enhanced potency to the CAR in tumor cell killing and cytokine responses.

[0369] In addition, by changing the length and composition of the CAR hinge (also known as the linker domain), for example, by replacing the CD8α-derived linker domain with a variable length TNFRSF19-derived domain or a domain derived from an immunoglobulin constant region and / or hinge, such as an IgG1-derived linker domain or an IgG4-derived linker domain incorporated into the CH2 and / or CH3 and / or hinge domain of an immunoglobulin molecule, or a modification thereof, the tumor antigen can be better brought into contact with the CAR binding domain. This is due to the fact that the appropriate length and flexibility of the CAR hinge / linker domain are necessary for optimal accessibility, binding to the tumor antigen, and CAR T cell activation.

[0370] In addition, the incorporation of tag molecules into the CAR construct sequence expressed on the surface of CAR T cells can be used for 1) identifying CAR T cells by flow cytometry during the manufacturing process and in clinical applications, 2) sorting / separating CAR T cells during the manufacturing process, 3) in the case of CAR-related toxicity, such as B cell aplasia in response to anti-CD19 CAR, cytokine release syndrome or CAR-related neurotoxicity, as a suicide tag to eliminate CAR T cells from the patient's body. For this purpose, the CAR construct sequence can include the truncated extracellular domain and transmembrane portion of a natural transmembrane protein (such as HER1 / EGFR, HER2 / Neu / erbB-2, NGFR / LNGFR / CD271, CD19, CD20 or other proteins). These or other sequence mimetic epitopes can also be used. By administering a clinical-grade antibody with tag reactivity, such as an antibody targeting EGFR (cetuximab), HER2 (trastuzumab), CD20 (rituximab) or other proteins, it is achieved to remove the labeled CAR T cells from the patient's circulation.

[0371] Figures 12A-12F Selected examples of the above CAR configurations are shown. The anti-CD33 CAR constructs described were designed using CD33 binding sequences derived from the immunoglobulin VH domain CD33_4, however, ScFv-format binder sequences can also be used.

[0372] Materials and methods:

[0373] (a) Establishment of chimeric antigen receptor (CAR)-expression vector

[0374] The CAR antigen binding domain sequence is derived from human anti-CD33 ScFv or heavy chain variable fragment. The binding sequence is connected to CD8a and a transmembrane domain (UniProt sequence ID P01732, aa 138-206), and then connected to 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signal transduction domain and CD3ζ signal transduction domain (CD247, aa52-163, reference sequence ID: NP_000725.1) frame to produce CAR T constructs. For some constructs, CD28 costimulatory sequences (UniProt ID: P10747, transmembrane domain, aa 153-179) are used instead of 4-1BB costimulatory sequences. In some constructs, CD8 connection and / or transmembrane domains are replaced with domains of various lengths derived from TNFRSF19 protein (UniProt ID: Q9NS68). For some sequences, a truncated epidermal growth factor receptor (tEGFR) tag (UniProt ID: P00533, various sequences) is incorporated into the CAR construct via a 2A peptide to enable in vitro labeling of transduced cells, as well as for use as a suicide switch for in vivo applications. The CAR construct sequence is cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Supernatants containing lentiviral vectors (LVs) are produced by transient transfection of HEK 293T cells, and the vectors are precipitated by centrifugation of the supernatant containing the lentiviral vectors and stored at -80 ° C.

[0375] Table 2 - Expression and Function Summary - Anti-CD33 CAR

[0376]

[0377]

[0378] Each application and patent cited herein, and each document or reference cited in each such application and patent (including during the pendency of each issued patent; the "application cited document"), and each PCT and foreign application or patent corresponding to and / or claiming priority from any of these applications and patents, and each document cited or referenced in each application cited document are hereby expressly incorporated herein by reference and may be used in the practice of the present invention. More generally, several documents or references are cited herein, whether in a reference list preceding the claims or in the text itself; and each of these documents or references (the "herein cited references") and each document or reference cited in each herein cited reference (including any manufacturer's instructions, guides, etc.) are hereby expressly incorporated herein by reference.

[0379] The foregoing description of some specific embodiments provides sufficient information so that other persons can easily modify or adjust such specific embodiments for different applications by applying current knowledge without departing from the general concept, and therefore such adjustments and modifications should and are intended to be included in the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for descriptive and not restrictive purposes. Exemplary embodiments have been disclosed in the drawings and the specification, and although specific terms may have been used, unless otherwise stated, they are used only in a general descriptive sense and not for limiting purposes, and the scope of the claims is therefore not limited thereto. In addition, those skilled in the art will understand that certain steps of the methods discussed herein can be sorted in an alternative order or the steps can be combined. Therefore, this means that the appended claims are not limited to the specific embodiments disclosed herein. Those skilled in the art will recognize or be able to determine many equivalents to the embodiments of the invention described herein using only routine experiments. Such equivalents are encompassed in the following claims.

[0380] References to sequence listings

[0381] This application contains a Sequence Listing that was submitted electronically to the United States Patent and Trademark Office as a PDF file entitled "Sequence Listing," which is incorporated by reference.

[0382] Sequence of the present disclosure

[0383] The nucleic acid and amino acid sequences listed below are shown using the standard letter abbreviations for nucleotide bases and the three-letter code for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. In the accompanying sequence listing:

[0384] SEQ ID NO: 1 Nucleotide sequence of CD33-reactive immunoglobulin heavy chain variable domain (VH-2)

[0385]

[0386] SEQ ID NO: 2 Amino acid sequence of CD33-reactive immunoglobulin heavy chain variable domain (VH-2)

[0387]

[0388] SEQ ID NO: 3 Nucleotide sequence of CD33-reactive immunoglobulin heavy chain variable domain (VH-4)

[0389]

[0390] SEQ ID NO: 4 Amino acid sequence of CD33-reactive immunoglobulin heavy chain variable domain (VH-4)

[0391]

[0392] SEQ ID NO: 5 Nucleotide sequence of CD33-reactive ScFv 9 binding domain

[0393]

[0394] SEQ ID NO:6 Amino acid sequence of CD33-reactive ScFv 9 binding domain

[0395]

[0396] SEQ ID NO: 7 Nucleotide sequence of CD33-reactive ScFv 10 binding domain

[0397]

[0398] SEQ ID NO: 8 Amino acid sequence of the CD33-reactive ScFv 10 binding domain

[0399]

[0400] SEQ ID NO: 9 Nucleotide sequence of CD33-reactive ScFv 12 binding domain

[0401]

[0402] SEQ ID NO: 10 Amino acid sequence of the CD33-reactive ScFv 12 binding domain

[0403]

[0404] SEQ ID NO: 11 Nucleotide sequence of CD33-reactive ScFv 15 binding domain

[0405]

[0406] SEQ ID NO: 12 Amino acid sequence of the CD33-reactive ScFv 15 binding domain

[0407]

[0408] SEQ ID NO: 13 Nucleotide sequence of leader / signal peptide sequence

[0409]

[0410] SEQ ID NO: 14 amino acid sequence of leader / signal peptide sequence

[0411]

[0412] SEQ ID NO: 15 Nucleotide sequence of LTG 1905_(EF1a-VH-2 CD33-CD8 TM-41BB-CD3ζ)

[0413]

[0414] SEQ ID NO: 16 Amino acid sequence of LTG 1905_(EF1a-VH-2 CD33-CD8 TM-41BB-CD3ζ)

[0415]

[0416] SEQ ID NO: 17 Nucleotide sequence of LTG 1906 (EF1a-VH-4 CD33-CD8 TM-41BB-CD3ζ) nucleic acid sequence

[0417]

[0418] SEQ ID NO: 18 Amino acid sequence of LTG 1906 (EF1a-VH-4 CD33-CD8 TM-41BB-CD3ζ)

[0419]

[0420] SEQ ID NO: 19 Nucleotide sequence of LTG 1936_(EF1a_ScFv9 CD33 CD8 TM-41BB-CD3ζ CAR)

[0421]

[0422] SEQ ID NO: 20 Amino acid sequence of LTG 1936_(EF1a_ScFv9 CD33 CD8 TM-41BB-CD3ζ)

[0423]

[0424] SEQ ID NO: 21 Nucleotide sequence of LTG 1937_(EF1a_ScFv10 CD33 CD8 TM-41BB-CD3ζ CAR)

[0425]

[0426]

[0427] SEQ ID NO: 22 Amino acid sequence of LTG 1937_(EF1a_ScFv10 CD33 CD8 TM-41BB-CD3ζ)

[0428]

[0429] SEQ ID NO: 23 Nucleotide sequence of LTG 1938_(EF1a_ScFv12 CD33 CD8 TM-41BB-CD3ζ)

[0430]

[0431] SEQ ID NO: 24 Amino acid sequence of LTG 1938_(EF1a_ScFv12 CD33 CD8 TM-41BB-CD3ζ)

[0432]

[0433] SEQ ID NO: 25 Nucleotide sequence of LTG 1939_(EF1a_ScFv15 CD33 CD8 TM-41BB-CD3ζ)

[0434]

[0435]

[0436] SEQ ID NO:26 Amino acid sequence of LTG 1939_(EF1a_ScFv15 CD33 CD8 TM-41BB-CD3ζ)

[0437]

[0438] SEQ ID NO: 27 Nucleotide sequence of DNA CD8 transmembrane domain

[0439]

[0440] SEQ ID NO: 28 Amino acid sequence of CD8 transmembrane domain

[0441]

[0442] SEQ ID NO: 29 Nucleotide sequence of DNA CD8 hinge domain

[0443]

[0444] SEQ ID NO: 30 Amino acid sequence of CD8 hinge domain

[0445]

[0446] SEQ ID NO: 31 Amino acid sequence of the hinge region from amino acids 118 to 178 of CD8α (NCBI RefSeq: NP.sub.--001759.3)

[0447]

[0448] SEQ ID NO: 32 Amino acid sequence of human IgG CL sequence

[0449]

[0450] SEQ ID NO: 33 Nucleotide sequence of 4-1BB DNA signaling domain

[0451]

[0452] SEQ ID NO: 34 Amino acid sequence of 4-1BB signaling domain

[0453]

[0454] SEQ ID NO: 35 Nucleotide sequence of CD3ζ DNA signaling domain

[0455]

[0456] SEQ ID NO: 36 Amino acid sequence of CD3ζ

[0457]

[0458] SEQ ID NO: 37 Nucleotide sequence of Scvf cd 19

[0459]

[0460] SEQ ID NO: 38 Amino acid sequence of Scvf cd 19

[0461]

[0462] SEQ ID NO: 39 Nucleotide sequence of GMCSF leader peptide

[0463]

[0464] SEQ ID NO: 40 Amino acid sequence of GMCSF leader peptide

[0465]

[0466] SEQ ID NO: 41 Nucleotide sequence of TNFRSF19 leader peptide

[0467]

[0468] SEQ ID NO: 42 Amino acid sequence of TNFRSF19 leader peptide

[0469]

[0470] SEQ ID NO: 43 Nucleotide sequence of CD8α leader peptide

[0471]

[0472] SEQ ID NO: 44 Amino acid sequence of CD8α leader peptide

[0473]

[0474] SEQ ID NO: 45 Nucleotide sequence of CD28 costimulatory domain

[0475]

[0476] SEQ ID NO: 46 Amino acid sequence of CD28 costimulatory domain

[0477]

[0478] SEQ ID NO: 47 Nucleotide sequence of CD3ζ activation domain

[0479]

[0480] SEQ ID NO: 48 Amino acid sequence of CD3ζ activation domain

[0481]

[0482] SEQ ID NO: 49 Nucleotide sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain is underlined)

[0483]

[0484] SEQ ID NO: 50 Amino acid sequence of TNFRSF19 hinge and transmembrane domain (transmembrane domain is underlined)

[0485]

[0486] SEQ ID NO: 51 Nucleotide sequence of TNFRSF19 transmembrane domain

[0487]

[0488] SEQ ID NO: 52 Amino acid sequence of the transmembrane domain of TNFRSF19

[0489]

[0490] SEQ ID NO: 53 Nucleotide sequence of TNFRSF19 hinge domain

[0491]

[0492] SEQ ID NO: 54 Amino acid sequence of the hinge domain of TNFRSF19

[0493]

[0494] SEQ ID NO: 55 Nucleotide sequence of truncated TNFRSF19 hinge domain

[0495]

[0496] SEQ ID NO: 56 Amino acid sequence of truncated TNFRSF19 hinge domain

[0497]

[0498] SEQ ID NO: 57 Nucleotide sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence is underlined)

[0499]

[0500] SEQ ID NO: 58 Amino acid sequence of the CD8a hinge domain fused to the TNFRSF19 transmembrane domain (transmembrane sequence is underlined)

[0501]

[0502] SEQ ID NO: 59 Nucleotide sequence of CD28 costimulatory domain

[0503]

[0504] SEQ ID NO: 60 Amino acid sequence of CD28 costimulatory domain

[0505]

[0506] SEQ ID NO: 61 Nucleotide sequence of CD3ζ form 2

[0507]

[0508] SEQ ID NO: 62 Amino acid sequence of CD3ζ version 2

[0509]

[0510] SEQ ID NO: 63 Furin P2A nucleotide sequence of furin

[0511]

[0512] SEQ ID NO: 64 Furin P2A amino acid sequence of furin (furin sequence is underlined)

[0513]

[0514] SEQ ID NO: 65 Nucleotide sequence of Furin T2A

[0515]

[0516] SEQ ID NO: 66 Amino acid sequence of Furin T2A (Furin sequence is underlined)

[0517]

[0518] SEQ ID NO: 67 Nucleotide sequence of truncated EGFR (tEGFR) tag

[0519]

[0520] SEQ ID NO: 68 Amino acid sequence of truncated EGFR (tEGFR) tag

[0521]

[0522] SEQ ID NO: 69 Nucleotide sequence of LTG1927 (EF1a-CD33_4-CD8 TM-CD28-CD3ζ-cfrag)

[0523]

[0524]

[0525] SEQ ID NO: 70 Amino acid sequence of LTG1927 (EF1a-CD33_4-CD8 TM-CD28-CD3ζ-cfrag)

[0526]

[0527] SEQ ID NO: 71 Nucleotide sequence of LTG_D0033 (Ef1a_CD33_4 VH TNFRSF19 H_TM_CD28z) nucleotide sequence

[0528]

[0529] SEQ ID NO: 72 Amino acid sequence of LTG_D0033 (Ef1a_CD33_4 VH TNFRSF19 H_TM_CD28z) nucleotide sequence

[0530]

[0531] SEQ ID NO: 73 Nucleotide sequence of LTG_D0034 (Ef1a_CD33_4 VH TNFRSF19 H_TM_4-1BBz) nucleotide

[0532]

[0533] SEQ ID NO:74 Amino acid sequence of LTG_D0034 (Ef1a_CD33_4 VH TNFRSF19 H_TM_4-1BBz)

[0534]

[0535] SEQ ID NO: 75 Nucleotide sequence of LTG_D0015 (Ef1a_CD33_4 VH CD8 BBz T2A tEGFR)

[0536]

[0537] SEQ ID NO:76 Amino acid sequence of LTG_D0015 (Ef1a_CD33_4 VH CD8 BBz T2A tEGFR)

[0538]

[0539] SEQ ID NO: 77 Nucleotide sequence of LTG_D0016 (Ef1a CD33_4 VH CD8 28z T2A tEGFR)

[0540]

[0541] SEQ ID NO:78 Amino acid sequence of LTG_D0016 (Ef1a CD33_4 VH CD8 28z T2A tEGFR)

[0542]

[0543] SEQ ID NO: 79 Nucleotide sequence of human IgG4 hinge

[0544]

[0545] SEQ ID NO: 80 Amino acid sequence of human IgG4 hinge

[0546]

[0547] SEQ ID NO: 81 Nucleotide sequence of human IgG4 CH2 domain

[0548]

[0549] SEQ ID NO: 82 Amino acid sequence of human IgG4 CH2 domain

[0550]

[0551] SEQ ID NO: 83 Nucleotide sequence of human IgG4 CH3 domain

[0552]

[0553] SEQ ID NO: 84 Amino acid sequence of human IgG4 CH3 domain

[0554]

[0555] SEQ ID NO: 85 Nucleotide sequence of human IgG4 hinge CH2 CH3 domain

[0556]

[0557] SEQ ID NO: 86 Amino acid sequence of human IgG4 hinge CH2 CH3 domain

[0558]

[0559] SEQ ID NO: 87 Nucleotide sequence of LTG_D0035 (Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z)

[0560]

[0561] SEQ ID NO: 88 Amino acid sequence of LTG_D0035 (Ef1a_CD33_4 VH H CH2 CH3 IgG4_CD8TM_CD28z)

[0562]

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus: (i) at least one extracellular antigen-binding domain, said extracellular antigen-binding domain comprising a CD33 antigen-binding domain consisting of a heavy chain variable region set forth in the amino acid sequence of any one of SEQ ID NOs: 2 or 4, or a single-chain variable fragment (ScFv) domain set forth in the amino acid sequence of any one of SEQ ID NOs: 6 or 12; (ii) a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: the α, β, or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; (iii) at least one costimulatory domain comprising a functional signaling domain selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137); and (iv) an intracellular signaling domain comprising a functional domain selected from the group consisting of 4-1BB (CD137), CD28, and CD3ζ signaling domains; The encoded extracellular CD33 antigen binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

2. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD33 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD33.

3. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD33 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD33.

4. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one CD33 antigen binding domain, at least one intracellular signaling domain, or both are connected to the transmembrane domain via a linker or spacer domain.

5. The isolated nucleic acid molecule of claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19 or CD28 and is connected to the transmembrane domain.

6. The isolated nucleic acid molecule of claim 1, wherein the leader nucleotide sequence consists of the nucleotide sequence shown below: SEQ ID NO: 13 encoding the leading amino acid sequence of SEQ ID NO: 14, or SEQ ID NO: 39 encoding the leading amino acid sequence of SEQ ID NO: 40, or SEQ ID NO: 41 encoding the leading amino acid sequence of SEQ ID NO: 42, or SEQ ID NO: 43 encoding the leading amino acid sequence of SEQ ID NO:

44.

7. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises the transmembrane domain of CD8.

8. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

9. The isolated nucleic acid molecule of claim 8, wherein the encoded at least one intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

10. The isolated nucleic acid molecule of claim 1, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

11. The isolated nucleic acid molecule of claim 10, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, or 4-1BB (CD137).

12. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1.

13. The CAR of claim 12, comprising at least one extracellular antigen binding domain, at least one transmembrane domain, at least one costimulatory domain, and at least one intracellular signal transduction domain, wherein the extracellular antigen binding domain comprises a CD33 antigen binding domain consisting of an amino acid sequence of any one of SEQ ID NO. 2, 4, 6, or 12.

14. The CAR of claim 13, wherein the CD33 antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD33.

15. The CAR of claim 13, wherein the CD33 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD33.

16. The CAR of claim 13, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising: the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

17. The CAR of claim 16, wherein the CD8 transmembrane domain consists of the amino acid sequence of SEQ ID NO:

27.

18. The CAR of claim 13, wherein the at least one extracellular antigen binding domain and the at least one intracellular signaling domain or both are connected to the transmembrane domain via a linker or a spacer domain, and the extracellular antigen binding domain comprises a CD33 antigen binding domain consisting of the amino acid sequence of any one of SEQ ID NO. 2, 4, 6 or 12.

19. The CAR of claim 18, wherein the linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, IgG4, or CD28 and is connected to the transmembrane domain.

20. The CAR of claim 15, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

21. The CAR of claim 20, wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137).

22. A vector comprising the nucleic acid molecule of claim 1.

23. The vector of claim 22, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, an adenovirus vector, a retrovirus vector, or a combination thereof.

24. The vector of claim 23, wherein the vector is a lentiviral vector.

25. The vector of claim 22, further comprising a promoter.

26. The vector of claim 25, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

27. A cell comprising the vector of claim 22.

28. The cell of claim 27, wherein the cell is a T cell.

29. The cell of claim 27, wherein the T cell is a CD8 + T cells.

30. The cell of claim 27, wherein the cell is a human cell.

31. A method for preparing cells, comprising transducing T cells with the vector of claim 22.

32. A method for producing an RNA-engineered cell population comprising introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule of claim 1.

33. Use of the cell of claim 27 in the preparation of a medicament for providing anti-tumor immunity in a mammal, wherein the mammal has leukemia.

34. Use of the CAR of claim 13 in the preparation of a medicament for treating or preventing cancer in a mammal, wherein the mammal has leukemia.

35. A pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population, wherein the T cells comprise the isolated nucleic acid molecule of any one of claims 1 to 11, the CAR of any one of claims 12 to 21, or the vector of any one of claims 22 to 26.

36. The pharmaceutical composition of claim 35, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

37. The pharmaceutical composition of claim 35, wherein the T cells are T cells from a human suffering from leukemia.

38. The pharmaceutical composition of claim 37, wherein the leukemia is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic T-cell leukemia (T-ALL), or acute lymphocytic B-cell leukemia (B-ALL).

39. Use of a pharmaceutical composition in the preparation of a medicament for treating a mammal suffering from leukemia, the pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise the isolated nucleic acid molecule of any one of claims 1 to 11, the CAR of any one of claims 12 to 21, or the vector of any one of claims 22 to 26, wherein the T cells are T cells of a subject suffering from cancer.

40. Use of a pharmaceutical composition for preparing a medicament for treating leukemia in a subject in need thereof, the pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise the isolated nucleic acid molecule of any one of claims 1 to 11, the CAR of any one of claims 12 to 21, or the vector of any one of claims 22 to 26, wherein the T cells are T cells of a subject suffering from cancer.

41. The use of claim 39 or 40, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising: the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

42. A method for producing a cell expressing a chimeric antigen receptor, the method comprising introducing into a cell the isolated nucleic acid molecule of claim 1, wherein the cell is a T cell or a population of cells comprising a T cell.

Images