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CN118284621A - Anti-FLT 3 antibodies, CARs, CAR T cells, and methods of use - Google Patents

Anti-FLT 3 antibodies, CARs, CAR T cells, and methods of use Download PDF

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CN118284621A
CN118284621A CN202280068117.6A CN202280068117A CN118284621A CN 118284621 A CN118284621 A CN 118284621A CN 202280068117 A CN202280068117 A CN 202280068117A CN 118284621 A CN118284621 A CN 118284621A
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cells
car
flt
acid sequence
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V·M·桑德勒
E·施雷斯塔
R·梁
C·R·希罗奇恩斯基
R·本杰胡达
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Hermogen Knicks Pharmaceutical Co ltd
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Hermogen Knicks Pharmaceutical Co ltd
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Abstract

Provided herein are anti-FLT 3 antibodies, or antigen binding fragments thereof, that include a heavy chain variable region, a light chain variable region, and single chain fragments (such as humanized anti-FLT 3 antibodies and fragments thereof). In some aspects, the antibody or fragment specifically binds human FLT3. Also provided herein are recombinant receptors, such as Chimeric Antigen Receptors (CARs), comprising such antibodies or fragments. Also provided herein are immune cells, such as CAR T cells, comprising such CARs. Also provided herein are methods of using such antibodies or fragments, CARs, and immune cells.

Description

Anti-FLT 3 antibodies, CARs, CAR T cells, and methods of use
Cross Reference to Related Applications
The present application claims the benefit of U.S. provisional patent application No. 63/233,530 filed 8/16 of 2021 and U.S. provisional patent application No. 63/253,009 filed 10/6 of 2021, each of which are incorporated herein by reference in their entirety.
Reference to electronic sequence Listing
The contents of the electronic sequence listing (HEPH _001_002WO_SeqList_ST26.Xml; size: 228,845 bytes; and date of creation: 2022, 8, 11 days) are incorporated herein by reference in their entirety.
Technical Field
In some aspects, the invention relates to anti-FLT 3 humanized antibodies or antigen binding fragments thereof, chimeric Antigen Receptors (CARs) comprising such antibodies or fragments, immune cells expressing such CARs, and uses of such antibodies, CARs, and cells.
Background
FLT3
FLT3 is Fms-associated receptor tyrosine kinase 3.FLT3 is also known as fetal liver kinase 2 (FLK 2). FLT3 is a member of the class III tyrosine kinase receptor family, is expressed in normal hematopoietic progenitor cells and leukemia blasts, and plays an important role in cell proliferation, differentiation and survival. Activation of FLT3 receptors by FLT3 ligands results in receptor dimerization and phosphorylation and activation of downstream signaling pathways, including the Janus kinase (JAK) 2 signal transducer (JAK 2), signal transducer and transcription activator (STAT) 5, and Mitogen Activated Protein Kinase (MAPK) pathways. Mutations in the FLT3 gene found in about 40% of AML patients are thought to promote their autophosphorylation and constitutive activation, resulting in ligand independent proliferation (Frankfurt O et al Current Opinion in Oncology (2007) 19 (6): 635-649).
Normal FLT3 expression is primarily limited to cd34+ Hematopoietic Stem Cells (HSCs), early hematopoietic progenitor cells (HPs), and Dendritic Cells (DCs). Normal differentiation of the downstream blood lineage is promoted by FLT3 activation that binds FLT3 ligand (FLT 3L).
FLT3 is highly expressed in a variety of hematological malignancies, including in most AML patients. AML blast cells of most AML patients express FLT3, and this expression is thought to promote survival and proliferation. Tyrosine Kinase Inhibitors (TKIs) have been developed to specifically target FLT3; however, secondary mutations that lead to resistance to FLT3 remain a major obstacle.
Hematopoietic stem cells
Hematopoietic stem cells are common progenitors of all blood cells. As pluripotent cells, they can differentiate into multiple cell lineages, but not all lineages are derived from three germ layers. Hematopoietic stem cells differentiate to produce lymphoid and myeloid lineages, which are the two major branches of hematopoiesis .(Kondo,M."Lymphoid and myeloid lineage commitmentin multipotent hematopoietic progenitors,"Immunol.Rev.2010Nov;238(1):37-46). lymphoid lineages including T cells, B cells, and Natural Killer (NK) cells. The myeloid lineage includes megakaryocytes and erythrocytes (MegE), and different subsets of granulocytes belonging to the myeloid lineage (neutrophils, eosinophils and basophils), monocytes, macrophages and mast cells (GM) (as above, reference Kondo M et al, Biology of hematopoietic stem cells and progenitors:implications for clinical application.Ann.Rev Immunol.2003;21:759-806.,Weissman IL.Translating stem and progenitor cell biology to the clinic:barriers and opportunities.Science(New York,NY.2000, 25, 2, and 287 (5457): 1442-6); see also Iwaskaki, h. And Akashi, k. "Myeloid lineage commitment from the hematopoietic stem cell," Immunity 26 (6) month 6 of 2007, 726-40).
HSCs have self-renewing potential and the ability to differentiate into the blood lineage; that is, when stem cells divide, on average 50% of the daughter cells form a cell lineage, while the remaining 50% do not differentiate. The process maintains the same number of stem cells through asymmetric cell division, such that each divided stem cell produces one new stem cell and one differentiated cell. In contrast, in symmetric division, stem cells produce 100% identical stem cells. (Gordon, M.stem cells and haemopsis, in. Hoffbrand, V., catovsky, D., tuddenham, E.G., 5 th edition Blackwell Publishing, (2005): DIFFERENTIAL NICHE AND WNT requirements during acute myeloid leukemia, pages 1-12 New York.).
Lymphoid lineages and myeloid lineages are separable at the progenitor cell level. Common lymphoid progenitor Cells (CLPs) can differentiate into all types of lymphocytes under physiological conditions without significant myeloid potential (Kondo M, SCHERER DC, miyamoto T, king AG, akashi K, sugamura K. Et al Cell-fate conversion of lymphoid committed progenitors by instructive actions of cytokines.Nature.2000, month 9, 21; 407 (6802): 383-6), although some myeloid-related genes may be detected in CLPs, depending on experimental conditions (Delogu A,Schebesta A,Sun Q,Aschenbrenner K,Perlot T,Busslinger M.Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells.Immunity.2006, month 3; 24 (3):269-81).
Similarly, common myeloid progenitor Cells (CMP) can produce all classes of myeloid cells, but there is no B cell potential or very low level for (Akashi K,Traver D,Miyamoto T,Weissman IL.A clonogenic common myeloid progenitor that givesrise to all myeloid lineages.Nature.2000, 3,9 days; 404 (6774):193-7). Another cell type, dendritic Cells (DCs), is not explicitly classified as lymphoid or myeloid lineages, as DCs can be derived from CLP or CMP(Manz MG,Traver D,Miyamoto T,Weissman IL,Akashi K.Dendritic cell potentials of early lymphoid and myeloid progenitors.Blood.2001, month 6, and 1; 97 (11) 3333-41; traver D, akashi K, manz M, merad M, miyamoto T, ENGLEMAN EG et al Development of CD8alpha-positive dendritic cells from a common myeloid progenitor.Science(New York,NY.2000, 12 months 15; 290 (5499):2152-4)). CMP can proliferate and differentiate into megakaryocyte-erythrocyte (MegE) progenitor cells and granulocyte-monocyte (GM) progenitor cells, which further produce megakaryocytes, erythrocytes, granulocytes, monocytes, and the like .(Iwasaki H,Akashi K.Myeloid lineage commitment from the hematopoietic stem cell.Immunity.2007;26:726-740).
The difference in the expression levels of transcription factors probably determines lineage assignment of differentiated cells. Transcription factors PU.1 and GATA-1 are associated with myeloid and erythroid/megakaryocyte lineage differentiation, respectively (Gordon, M.stem cells and haemopsis, in. Hoffbrand, V., catovsky, D., tuddenham, E.G., 5 th edition Blackwell Publishing, (2005): DIFFERENTIALNICHE AND WNT requirements during acute myeloid leukemia, pages 1-12 New York.)
Characterization of HSC
HSCs are undifferentiated and resemble small lymphocytes. Most HSCs are quiescent, in the G0 phase of the cell cycle, which protects them from cell cycle dependent drugs. The resting state of stem cells is maintained by transforming growth factor-beta (TGF-beta). The activity of TGF- β is mediated by p53, p53 being a tumor suppressor gene that regulates cell proliferation and targets cyclin-dependent kinase inhibitor p21 (Gordon, m. Stem cells and haemo poisis. In: hoffbrand, v., catovsky, d., tuddenham, e.g., 5 th edition Blackwell Publishing, (2005): DIFFERENTIAL NICHE AND WNT requirementsduring acute myeloid leukemia, pages 1-12 New york.). Quiescence of HSCs is critical not only for long-term preservation of stem cell compartments and maintenance of stem cell banks, but also for minimizing accumulation of replication-related mutations. Many intrinsic transcription factors that maintain HSC quiescence are found to be associated with leukemia. For example, chromosomal translocations leading to fusion of FoxO and myeloid/lymphoid leukemia or mixed lineage leukemia have been reported in acute myeloid leukemia (see, e.g., blocks Sergio Paulo Bydlowski and Felipe de Lara Janz(2012).Hematopoietic Stem Cell in Acute Myeloid Leukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo(), ISBN: 978-953-307-930-1.
Most normal HSCs were present in the CD34+/CD38-/CD90+ bone marrow cell fraction, and some HSCs were also observed in CD 34-/Lin-cells. The CD34+/CD38+ cell fraction contains some HSCs with short term repopulating activity. Other recognized markers include the tyrosine kinase receptor c-kit (CD 117) and the deficiency of terminally differentiated markers such as CD4 and CD8 (Rossi et al Methods in Molecular Biology (2011) 750 (2): 47-59).
Classification of HSC
Hematopoietic stem cell banks can be subdivided into three major classes: (1) Short term HSCs, which can only generate clones of cells within 4-6 weeks; (2) Mid-term HSCs capable of maintaining differentiated cell offspring for 6-8 months prior to extinction; and (3) long term HSCs capable of maintaining hematopoiesis indefinitely. (Testa U.S. Annals of therapeutics (2011) 90 (3): 245-271).
Hematopoiesis
Hematopoiesis is a highly coordinated process in which HSCs differentiate into mature blood cells supported by a specialized regulatory microenvironment consisting of components that control stem and progenitor cell fate specification and maintain their development by providing the necessary factors ("niches"). The term "Bone Marrow (BM) niche" as used herein refers to a well-organized structure composed of elements (e.g., osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes, and extracellular matrix proteins (ECM)) that play an important role in survival, growth, and differentiation of different blood cell lineages. The bone marrow niche is an important postnatal microenvironment in which HSCs proliferate, mature and produce myeloid and lymphoid progenitor cells.
Bone Marrow (BM) is present in the medullary cavity of all animal bones. It consists of a variety of precursor cells and mature cell types, including hematopoietic cells (precursors to mature blood cells) and stromal cells (precursors to extensive connective tissue cells), both of which appear to be capable of differentiating into other cell types. The mononuclear fraction of bone marrow contains stromal cells, hematopoietic precursor cells, and endothelial precursor cells.
Unlike secondary lymphoid organs (such as the spleen) that have unique macroscopic structures (including red and white marrow), BM has no defined structural features (except for the endosteal membranes that contain osteoblasts). The endosteal region is in contact with calcified hard bone and provides the special microenvironment necessary to maintain HSC activity (Kondo M, immunology Reviews (2010) 238 (1): 37-46; bydlowski and de Lara Janz(2012)).Hematopoietic Stem Cell in Acute Myeloid Leukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo(, code) ISBN: 978-953-307-930-1).
Within the niche, HSCs are thought to receive support and growth signals originating from a number of sources, including: fibroblasts, endothelial cells and reticulocytes, adipocytes, osteoblasts and Mesenchymal Stem Cells (MSCs). The primary function of the niche is to integrate local changes in nutrient, oxygen, paracrine and autocrine signals and alter the quiescence, transport and/or expansion of HSCs in response to signals from the systemic circulation (Broner, F. & Carson, mc. Topics in bone biology. Springer.2009;4: pages 2-4 New York, usa.).
Although the nature of real MSCs remains misunderstood, it has recently been reported that CD146 MSCs expressing CXC chemokine ligand 12 (CXCL 12) are self-renewing progenitor cells that reside on the surface of the blood sinuses and contribute to the tissue of the wall structure of the blood sinuses, producing angiopoietin-1 (Ang-1), and capable of generating osteoblasts that form endosteal niches (Konopleva, MY, & Jordan, CT, biology and Therapeutic Targeting (2011) 9 (5): 591-599). These CXCL12 reticulocytes can serve as a transport pathway for shuttling HSCs between the osteoblast niche and the vascular niche, providing the necessary but different maintenance signals in the niche.
Cytokines and chemokines produced by bone marrow MSCs are concentrated in specific niches secondary to different local production and by the action of glycosaminoglycans that bind the cytokines. In these CXCL 12/stromal cell derived factor-1α positively regulates HSC homing, while the transforming growth factor FMS-like tyrosine kinase 3 (Flt 3) ligand and Ang-1 act as resting factors (see, e.g., accession number Sergio Paulo Bydlowski and Felipe de Lara Janz(2012).Hematopoietic Stem Cell in Acute Myeloid Leukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo(), ISBN: 978-953-307-930-1). CXCL12-CXCR4 signaling is involved in HSC homing into BM during the ontogenesis, survival and proliferation of colony forming progenitor cells. CXCR4 selective antagonist-induced mobilization of HSCs into peripheral blood further suggests a role for CXCL12 in preserving HSCs in hematopoietic organs. BM implantation involves subsequent intercellular interactions through the complex extracellular matrix produced by BMSCs. Thus, vascular cell adhesion molecule-1 (VCAM-1) or fibronectin is critical for adhesion to BM-derived MSCs. In this way, control of the kinetics of hematopoietic stem cell proliferation is critical to the regulation of correct hematopoietic cell production. These control mechanisms can be categorized as either intrinsic or extrinsic to the stem cells, or a combination of both (see, e.g., sergio Paulo Bydlowski and Felipe de Lara Janz(2012).Hematopoietic Stem Cell in Acute MyeloidLeukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo(, et seq.) ISBN: 978-953-307-930-1).
Self-renewal and differentiation of HSCs may be controlled by external factors (external control), such as intercellular interactions or cytokines in the hematopoietic microenvironment, such as SCF (stem cell factor) and its receptors c-kit, flt-3 ligand, TGF- β, TNF- α, etc. Cytokines regulate a variety of hematopoietic cell functions by activating a variety of signal transduction pathways. The major pathways involved in cell proliferation and differentiation are the Janus kinase (Jak)/Signal Transducer and Activator of Transcription (STAT), mitogen Activated Protein (MAP) kinase and Phosphatidylinositol (PI) 3-kinase pathways (Sergio Paulo Bydlowski and Felipe de LaraJanz(2012).Hematopoietic Stem Cell in Acute Myeloid Leukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo(, code) and ISBN: 978-953-307-930-1.
In addition, other transcription factors such as Stem Cell Leukemia (SCL) hematopoietic transcription factors; GATA-2; and expression of gene products involved in cell cycle control, such as cyclin-dependent kinase inhibitors (CKI) pl6, p21 and p27, have been shown to be critical for the development of hematopoietic cells from the earliest stage (intrinsic control) (Sergio Paulo Bydlowski and Felipede Lara Janz(2012).Hematopoietic Stem Cell in Acute MyeloidLeukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo(, code) ISBN: 978-953-307-930-1.
The Notch-1-Jagged pathway may be used to integrate extracellular signaling with intracellular signaling and cell cycle control. Notch-1 is a surface receptor on hematopoietic stem cell membranes that binds to ligand Jagged on its stromal cells. This results in cleavage of the cytoplasmic portion of Notch-1, which can then act as a transcription factor (Gordon, m. Stem cells and haemopsis. In: hoffbrand, v., catovsky, d., tuddenham, e.g., 5 th edition Blackwell Publishing, (2005): DIFFERENTIAL NICHE AND WNT requirements during acute myeloid leukemia, pages 1-12 New york.).
Disorders treated with BM/HSC transplantation
Disorders treated using Bone Marrow (BM)/Hematopoietic Stem Cell (HSC) transplantation include, but are not limited to, acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), chronic Myeloid Leukemia (CML), maternal plasmacytoid dendritic cell tumor (BPDCN), peripheral T cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, hodgkin's lymphoma (Hodgkin's lymphoma), non-Hodgkin's lymphoma, neuroblastoma, non-malignant hereditary disorders, and acquired myelopathy (e.g., sickle cell anemia, severe beta thalassemia, refractory Diamond-black fan anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia, paroxysmal sleep hemoglobinuria, pure red cell aplastic anemia, fanconi anemia, megakaryocytopenia, or congenital thrombocytopenia), multiple myeloma, and Severe Combined Immunodeficiency (SCID).
Malignant tumor of hematopoietic system
Most hematopoietic malignancies contain functionally heterogeneous cells, only a subset of which (called cancer stem cells) are responsible for tumor maintenance. Cancer stem cells are so named because they have qualities similar to normal tissue stem cells, including self-renewal, prolonged survival, and the ability to produce cells with more differentiated properties (Jones RJ and Armstrong SA, biol Blood Marrow Transplant.20088 Jan;14 (supply 1): 12-16).
Transformation events in hematopoietic stem cells can produce several different malignancies, including but not limited to chronic myeloid leukemia, myelodysplastic syndrome, acute myeloid leukemia, and possibly even acute lymphoblastic leukemia, depending on the degree of differentiation associated with oncogenic stroke (Jones RJ and Armstrong SA, biol Blood Marrow Transplant.2008Jan;14 (support 1): 12-16).
The cancer stem cell concept is based on the following ideas: tumors of a particular tissue generally appear to "try" to reproduce the cellular heterogeneity present in the tissue of origin, and thus there are stem cell-like cells present in the tumor that produce different types. The basic test of this hypothesis is whether tumor cells can be divided into cells with tumor regeneration capability and cells without such capability. This cellular hierarchy is most clearly demonstrated in acute myelogenous leukemia, some of which possess cells with unique immunophenotypes that are capable of eliciting leukemia in immunodeficient mice, while most cells are incapable of eliciting the development of leukemia. In addition, leukemic-initiating cells also produce cells that lose tumor initiating activity, thus reproducing the cellular heterogeneity that exists in the original tumor (Lapidot T et al, nature.1994;367:645-648; bonnet D et al, nat Med.1997; 3:730-737).
Acute myeloid leukemia
Acute Myeloid Leukemia (AML) is a clonal disorder characterized by a arrest in myeloid lineage differentiation accompanied by accumulation of immature progenitor cells in the bone marrow, leading to hematopoietic failure (Poll yea DA et al, british Journal ofHaematology (2011) 152 (5): 523-542). There is a wide range of inter-patient heterogeneity in the appearance of leukemic blast cells. The discovery of leukemia initiating cells in Acute Myeloid Leukemia (AML) began with the discovery that most AML blast cells did not proliferate and only a small fraction was able to form new colonies (Testa U, annals of Hematology (2011) 90 (3): 245-271). A common feature of all AML cases is an abnormal differentiation arrest, resulting in an accumulation of more than 20% of blast cells in the bone marrow (Gilliland, DG and TALLMAN MS, CANCER CELL (2002) 1 (5): 417-420).
Over 80% of myeloid leukemia is associated with at least one chromosomal rearrangement (Pandolfi PP, oncogene (2001) 20 (40): 5726-5735), and over 100 different chromosomal translocations have been cloned (gillliland, DG and TALLMAN MS, CANCER CELL (2002) 1 (5): 417-420). These translocations often involve genes encoding transcription factors that have been shown to play an important role in hematopoietic lineage development. Thus, the change in transcription machinery appears to be a common mechanism leading to differentiation arrest (Pandolfi PP, oncogene (2001) 20 (40): 5726-5735;Tenen DG,Nature Reviews of Cancer (2003) 3 (2): 89-101).
Clinical studies and experimental animal models indicate that at least two genetic alterations are required for the clinical manifestation of acute leukemia. The cooperation between the class I activating mutations and the class II mutations inducing termination of differentiation resulted in AML according to the model proposed by Gilliland & Tallman (CANCER CELL (2002) 1 (5): 417-420). Class I mutations (such as mutations in receptor tyrosine kinase genes FLT3 and KIT, RAS family members, and loss of function of neurofibrin 1) confer a proliferative and/or survival advantage to hematopoietic progenitor cells, which is typically the result of aberrant activation of signal transduction pathways. Class II mutations result in a stop of differentiation via interference with transcription factors or coactivators (Frankfurt O et al Current Opinion in Oncology (2007) 19 (6): 635-649). Although Leukemia Stem Cells (LSCs) appear to share many cell surface markers previously identified for HSCs, such as CD34, CD38, HLA-DR, and CD71, several groups reported surface markers that were differentially expressed in the two populations. For example, CD90 or Thy-1 has been described as potentially specific for LSC compartments. Thy-1 is down-regulated in normal hematopoiesis as the most primitive stem cells progress to the progenitor stage. (Hope KJ et al, architecture of MEDICAL RESEARCH (2003) 34 (6): 507-514).
The interaction between CXCL12 (stromal cell derived factor-1 a) and its receptor CXCR4 on leukemic progenitors helps them to home to the bone marrow microenvironment. CXCR4 levels were significantly elevated in leukemia cells from AML patients, and CXCR4 expression was correlated with poor outcome (Konopleva MY and Jordan CT, biology and Therapeutic Targeting (2011) 29 (5): 591-599).
Constitutive activation of primary human AML stem cell nuclear factor kappa f3 (NF-kβ) pathway I provides evidence that NF-kβ plays an important role in the overall survival of LSCs as well as general AML cell types. (Konopleva MY and Jordan CT, biology and Therapeutic Targeting (2011) 29 (5): 591-599).
AML patients have a poor clinical prognosis and limited treatment options, hematopoietic Stem Cell Transplantation (HSCT) following bone marrow cell depletion is the only curative treatment. Common conditioning protocols indiscriminately kill all highly proliferating cell types, leading to life threatening side effects, and may also be ineffective for resting AML subpopulations.
Lymphoid malignancies
When cells enter the normal differentiation stage, the self-renewal capacity of most tissues is lost; for example, myeloid lineage blood cells that exceed hematopoietic stem cell levels no longer have self-renewal capacity. A notable exception to the loss of self-renewal associated with differentiation is the lymphoid system, where self-renewal capacity is maintained up to the memory lymphocyte stage in order to maintain lifelong immunological memory (Fearon DT et al, science.2001;293:248-250; luckey CJ et al, proc NATL ACAD SCI US A.2006; 103:3304-3309). Somatic hypermutations serve as markers for the differentiation stages of B cell malignancy. Generally, the presence of somatic hypermutations identifies a tumor as occurring in a germinal center B cell or post germinal center B cell, while the absence of mutations identifies a pre germinal center B cell. In contrast to myeloid malignancies, but consistent with the self-renewing capacity retained by the lineage, immunoglobulin (lg) mutation patterns suggest that B-cell malignancies may be caused by cells throughout the B-cell differentiation stage (Lapidot T et al, nature 1994;367:645-648; bonnet D and Dick JE, nat Med 1997;3:730-737; jones RJ et al, J NATL CANCER Inst 2004; 96:583-585).
Multiple myeloma
Multiple Myeloma (MM) is generally considered a malignant plasma cell disease, many of the clinical consequences of which are caused by plasma cell mass (PLASMA CELL bulk). However, normal plasma cells are terminally differentiated and lack self-renewing capacity, and it has been clear for more than 30 years that only a few cells from mouse and human MMs have clonogenic capacity. These rare colony forming cells are known as "tumor stem cells" (Park CH et al, J NATL CANCER Inst.1971;46:411-422; hamburger AW and Salmon SE, science.1977; 197:461-463). MM plasma cells are derived from a small population of self-renewing cancer stem cells similar to memory B cells. These clonotype B cells circulate not only in most patients, but also are resistant to many standard anti-MM drugs and thus appear to be responsible for most disease recurrence (Matsui WH et al blood.2004;103:2332-2336; kukreja A et al J Exp Med.2006;203:1859-1865; jones RJ and Armstrong SA, biol Blood Marrow Transplant.20088 Jan;14 (supplement 1): 12-16).
Hodgkin lymphoma
Reed-Sternberg (RS) cells are hallmarks of Hodgkin's Lymphoma (HL), the only blood cells that occasionally express CD138 in addition to plasma cells (carbon A et al, blood 1998; 92:2220-2228). HL Cell lines have been demonstrated to include a small population of cells that lack the RS markers CD15 and CD30 present on the remaining cells, while expressing markers consistent with the memory B Cell phenotype (Newcom SR et al, int J Cell cloning.1988;6:417-431; jones RJ et al, blood.2006; 108:470). This small subset of phenotypic memory B cells has all clonogenic capacity within the HL cell line. Most HL patients, including those suffering from early stage disease, contain circulating memory B cells with the same clonal lg gene rearrangement as the patient RS cells (Jones RJ et al, blood.2006;108:470; jones RJ and Armstrong SA, biol Blood Marrow Transplant.20088 Jan;14 (supply 1): 12-16). These data indicate that these clonotype memory B cells are likely to represent HL stem cells.
Treatment of hematological malignancies
Hematopoietic Stem Cells (HSC) are used in bone marrow transplantation to treat hematological malignancies and non-malignant conditions (Warner et al, oncogene (2004) 23 (43): 7164-7177). Bone Marrow (BM) was transplanted as an unfractionated pool of cells for many years (see, e.g., the Sergio Paulo Bydlowski and Felipe de Lara Janz(2012).Hematopoietic Stem Cell in Acute Myeloid Leukemia Development,Advances in Hematopoietic Stem Cell Research,Dr.Rosana Pelayo( editions), ISBN:978-953-307-930-l, before researchers found which cellular components were responsible for donor hematopoiesis and implantation of the immune system in bone marrow ablated patients. Preparation or conditioning of a patient for bone marrow/hematopoietic stem cell (BM/HSC) transplantation is a key element of the procedure. It has two main purposes: (1) It provides adequate immunosuppression for the patient and clears sufficient niche space in the bone marrow for transplanted HSCs, allowing transplanted cells to engraft in the recipient; (2) it generally helps eradicate the source of malignancy.
Traditionally, patient conditioning has been achieved by administering a maximum tolerated dose of a mixture of chemotherapeutic agents with or without radiation. The components of the mixture are typically selected to have non-overlapping toxicities. All the preparation schemes currently in use are toxic and have serious side effects that can be life threatening. These side effects include mucositis, nausea and vomiting, hair loss, diarrhea, rash, peripheral neuropathy, infertility, pulmonary toxicity and hepatotoxicity. Many of these side effects are particularly dangerous for elderly and ill patients and often become determining factors in determining whether a patient is to receive a transplant.
Thus, there is a need to prepare or condition patients suitable for bone marrow/hematopoietic stem cell (BM/HSC) transplantation in the absence of these toxicities.
There is also a need to treat hematological malignancies, such as AML, in the absence of these toxicities.
Disclosure of Invention
In some aspects, the disclosure provides a humanized antibody or antigen-binding fragment thereof that binds (such as specifically binds) human and rhesus FLT 3. In some aspects, the disclosure provides a humanized antibody or antigen-binding fragment thereof that binds (such as specifically binds) human FLT 3.
In some embodiments, the disclosure provides an anti-FLT 3 humanized antibody or antigen binding fragment thereof, wherein the antibody or fragment comprises a light chain variable region (VL) comprising amino acid sequence ：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID NO 38 having at least 95% identity to any one of the sequences selected from the group consisting of SEQ ID NOs.
In some embodiments, the disclosure provides an anti-FLT 3 humanized antibody or antigen binding fragment thereof, wherein the antibody or fragment comprises a heavy chain variable region (VH) comprising amino acid sequence ：SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO;24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID No. 27 having at least 95% identity to any one of the sequences selected from the group consisting of SEQ ID NOs.
In some embodiments, the disclosure provides an anti-FLT 3 humanized antibody or antigen binding fragment thereof, wherein the antibody or fragment comprises:
(i) A light chain variable region (VL) comprising an amino acid sequence ：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 having at least 95% identity to any one of the sequences selected from the group consisting of SEQ ID No. 38; and/or
(Ii) A heavy chain variable region (VH) comprising an amino acid sequence ：SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO;24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID No. 27 having at least 95% identity to any one of the sequences selected from the group consisting of SEQ ID NOs.
In any of the foregoing or related aspects and embodiments, the VL comprises the amino acid sequence of SEQ ID No. 1 and the VH comprises the amino acid sequence of SEQ ID No. 3. In any of the foregoing or related aspects and embodiments, the VL comprises the amino acid sequence of SEQ ID No. 2 and the VH comprises the amino acid sequence of SEQ ID No. 3.
In any of the foregoing or related aspects and embodiments, the disclosure provides an anti-FLT 3 humanized antibody or fragment thereof, wherein the VL comprises Complementarity Determining Regions (CDRs) that are at least 97%, 98%, 99% or 100% identical to the amino acid sequences of CDR-L1 of SEQ ID No. 86, CDR-L2 of SEQ ID No. 87, and CDR-L3 of SEQ ID No. 88. In some of these embodiments, the CDRs are determined by Kabat.
In any of the foregoing or related aspects and embodiments, the disclosure provides an anti-FLT 3 humanized antibody or fragment thereof, wherein the VH comprises CDRs having at least 97%, 98%, 99% or 100% identity to the amino acid sequence of CDR-H1 of SEQ ID No. 89, CDR-H2 of SEQ ID No. 90, and CDR-L3 of SEQ ID No. 91. In some of these embodiments, the CDRs are determined by Kabat.
In any of the foregoing or related aspects and embodiments, the disclosure provides an anti-FLT 3 humanized antibody or fragment thereof, wherein (i) the VL comprises Complementarity Determining Regions (CDRs) that have at least 97%, 98%, 99% or 100% identity to the amino acid sequences of CDR-L1 of SEQ ID NO:86, CDR-L2 of SEQ ID NO:87, and CDR-L3 of SEQ ID NO: 88; and (ii) the VH comprises a CDR which has at least 97%, 98%, 99% or 100% identity to the amino acid sequences of CDR-H1 of SEQ ID NO:89, CDR-H2 of SEQ ID NO:90 and CDR-L3 of SEQ ID NO: 91. In some of these embodiments, the CDRs are determined by Kabat.
In any of the foregoing or related aspects and embodiments, the antigen-binding fragment of a humanized anti-FLT 3 antibody described herein is a single chain variable domain (scFv) (such as a scFv comprising any VH and any VL described herein or mentioned in the foregoing aspects and embodiments). In some embodiments, the scFv comprises (or consists essentially of, or consists of) an amino acid sequence selected from the group comprising: SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49. In some embodiments, the scFv comprises (or consists essentially of, or consists of) an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 4. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO. 5. In some embodiments, the scFv comprises a linker between VL and VH, wherein the linker is of formula (Gly 3-4-Ser)1-4. In some embodiments, the scFv comprises a linker between VL and VH, wherein the linker is GGGGSGGGGSGGGSGGGGS (SEQ ID NO: 53).
In any of the foregoing or related aspects and embodiments, the anti-FLT 3 antibodies and fragments thereof (e.g., scFv) described herein do not compete (or substantially do not compete) with FLT3 ligands for binding to FLT3.
In some aspects, the disclosure provides nucleic acids encoding any of the anti-FLT 3 antibodies and antigen binding fragments (e.g., scFv) described herein. In some aspects, the disclosure provides a vector comprising a nucleic acid encoding any of the anti-FLT 3 antibodies and antigen binding fragments (e.g., scFv) described herein. In some aspects, the disclosure provides a recombinant receptor (e.g., chimeric antigen receptor) comprising any of the anti-FLT 3 antigen binding fragments (e.g., scFv) described herein. In some aspects, the disclosure provides nucleic acids encoding recombinant receptors (e.g., chimeric antigen receptors) comprising any anti-FLT 3 antigen binding fragment (e.g., scFv) described herein. In some aspects, the disclosure provides a vector comprising a nucleic acid encoding a recombinant receptor comprising any of the anti-FLT 3 antigen binding fragments (e.g., scFv) described herein.
In some aspects, the present disclosure provides a Chimeric Antigen Receptor (CAR), wherein the CAR comprises: (i) An extracellular domain comprising (a) an antibody or fragment of any of the foregoing or related aspects, or (b) an anti-FLT 3 scFv of any of the foregoing or related aspects and embodiments; (ii) a transmembrane domain; and (iii) an intracellular domain.
In any of the foregoing or related aspects and embodiments of the CAR, the transmembrane domain is a CD3 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, or a CD28 transmembrane domain. In some embodiments of the CAR, the transmembrane domain is a CD8 transmembrane domain (e.g., a CD8 a transmembrane domain).
In any of the foregoing or related aspects and embodiments of the CAR, the intracellular domain comprises an activation domain (e.g., wherein the activation domain transmits an activation signal after the extracellular domain binds FLT3 when the CAR is expressed in a T cell). In some embodiments, the disclosure provides a CAR, wherein the activation domain (in the intracellular domain) comprises an intracellular signaling domain of cd3ζ, cd3ε, or fcrγ. In some embodiments, the disclosure provides a CAR comprising a cd3ζ activation domain/intracellular signaling domain. In some embodiments of the CAR, the intracellular domain further comprises one or more co-stimulatory domains. In some embodiments, the one or more co-stimulatory domains is from one or more of: CD28, 4-1BB, CD27, OX40 or ICOS. In some embodiments, one or more co-stimulatory domains is from CD28 and/or 4-1BB.
In any of the foregoing or related aspects and embodiments of the CAR, the CAR comprises a spacer or hinge region between the extracellular domain and the transmembrane domain. In some embodiments, the spacer or hinge region is from the extracellular domain of CD8 (e.g., CD8 a).
In any of the foregoing or related aspects and embodiments of the CAR, the extracellular domain further comprises a cleavable signal peptide.
In any of the foregoing or related aspects and embodiments of the CAR, the extracellular domain comprises an scFv comprising the amino acid sequence of SEQ ID No. 4; the transmembrane domain comprises a CD8 transmembrane domain; and the intracellular domain comprises an intracellular signaling domain of cd3ζ and a costimulatory domain of CD28 and/or 4-1 BB.
In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) an amino acid sequence selected from the group comprising: SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 6. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 9. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 10. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 11. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 12. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 13. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 14. In some embodiments, a CAR described herein comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO:15.
In any of the foregoing or related aspects and embodiments of the CAR, the CAR further comprises a safety switch polypeptide (e.g., wherein the safety switch polypeptide binds to the CAR through a self-cleaving peptide). In some embodiments, the safety switch polypeptide is iCasp9 or EGFRt. In some embodiments, the self-cleaving peptide is T2A, P2A, E2A, F a or IRES. In some embodiments, the self-cleaving peptide is T2A.
In any of the foregoing or related aspects and embodiments of the CAR, when the extracellular domain binds to FLT3 (e.g., on the surface of a cancer cell, hematopoietic stem cell, hematopoietic progenitor cell, or dendritic cell), the immune cell (e.g., T cell) expressing the CAR is activated or stimulated to proliferate. In some embodiments, when the CAR is expressed on the surface of an immune cell (e.g., a T cell), it directs the immune cell to kill cells that express FLT 3.
In some aspects, the disclosure provides an immune cell (e.g., T cell) or population of immune cells (e.g., T cells) that expresses a CAR of any of the foregoing or related aspects and embodiments. In some aspects, the disclosure provides an immune cell (e.g., T cell) or population of immune cells (e.g., T cells) comprising a nucleic acid encoding a CAR of any of the foregoing or related aspects and embodiments. In any of the foregoing or related aspects and embodiments, the immune cell is a T cell, NK cell, macrophage or monocyte. In some embodiments, the immune cell is a T cell.
In any of the foregoing or related aspects and embodiments, the immune cell (e.g., T cell) comprises a nucleic acid, wherein the nucleic acid comprises a sequence selected from the group consisting of: SEQ ID NO. 60, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68 and SEQ ID NO. 69.
In any of the foregoing or related aspects and embodiments, the immune cells (e.g., T cells) have been derived from a subject (e.g., human) prior to introducing the CAR or nucleic acid. In some embodiments, the immune cells expressing the CAR or comprising the nucleic acid are further expanded to generate a population of immune cells.
In some embodiments, any anti-FLT 3 CAR described herein is cytotoxic to AML cells in vitro.
In some embodiments, any immune cell described herein is characterized by stable expression of any anti-FLT 3 CAR described herein.
In some embodiments, any immune cell expressing an anti-FLT 3 CAR described herein is characterized by high proliferative potential.
In some aspects, the present disclosure provides a pharmaceutical composition comprising (i) a humanized anti-FLT 3 antibody or fragment of any one of the preceding or related aspects and embodiments, and (ii) a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a pharmaceutical composition comprising: (i) The scFv of any one of the preceding or related aspects and embodiments, and (ii) a pharmaceutically acceptable carrier.
In some aspects, the present disclosure provides a pharmaceutical composition comprising (i) an immune cell (e.g., a T cell) of any of the preceding or related aspects and embodiments, and (ii) a pharmaceutically acceptable carrier.
In some aspects, the present disclosure provides a pharmaceutical composition comprising (i) a population of immune cells (e.g., T cells) of any one of the preceding or related aspects and embodiments, and (ii) a pharmaceutically acceptable carrier.
In some aspects, the present disclosure provides a method of treating hematological cancer in a subject in need thereof, wherein the method comprises administering to the subject (e.g., a therapeutically effective amount of): (i) The humanized anti-FLT 3 antibody or fragment (e.g., scFv) of any one of the preceding or related aspects and embodiments, or (ii) a pharmaceutical composition comprising such a humanized anti-FLT 3 antibody or fragment (e.g., scFv).
In some aspects, the present disclosure provides a method of treating hematological cancer in a subject in need thereof, wherein the method comprises administering to the subject (e.g., a therapeutically effective amount of): (i) Immune cells (e.g., T cells) of any of the foregoing or related aspects and embodiments (such as cells expressing any of the CARs described herein), (ii) immune cells (e.g., T cells) of any of the foregoing or related aspects and embodiments (such as cells expressing any of the CARs described herein), or (ii) a pharmaceutical composition of such immune cells or immune cell populations.
In some aspects, the present disclosure provides methods for preparing or conditioning a subject in need thereof for hematopoietic cell transplantation, wherein the methods comprise administering to the subject (e.g., a therapeutically effective amount): (i) The humanized anti-FLT 3 antibody or fragment (e.g., scFv) of any one of the preceding or related aspects and embodiments, or (ii) a pharmaceutical composition comprising such a humanized anti-FLT 3 antibody or fragment (e.g., scFv).
In some aspects, the present disclosure provides methods for preparing or conditioning a subject in need thereof for hematopoietic cell transplantation, wherein the methods comprise administering to the subject (e.g., a therapeutically effective amount): (i) Immune cells (e.g., T cells) of any of the foregoing or related aspects and embodiments (such as cells expressing any of the CARs described herein), (ii) populations of immune cells of any of the foregoing or related aspects and embodiments (such as cells expressing any of the CARs described herein), or (ii) pharmaceutical compositions of such immune cells or populations of immune cells.
In any of the foregoing or related aspects and embodiments of the conditioning methods, the method further comprises administering to the subject a hematopoietic cell transplant after administration. In some embodiments, hematopoietic cell transplantation comprises transplanting hematopoietic stem cells and/or hematopoietic progenitor cells to the subject. In some embodiments, the hematopoietic cell transplantation is performed from 5 days to 6 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed about 2 to 3 weeks after administration.
In any of the foregoing or related aspects and embodiments, the hematological cancer is Acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Myeloid Leukemia (CML), chronic Lymphoblastic Leukemia (CLL), blast plasmacytoid dendritic cell tumor (BPDCN), peripheral T cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, non-malignant hereditary or acquired bone marrow disorder, multiple myeloma or dendritic cell tumor. In some embodiments, the hematological cancer is AML. In some embodiments, the hematological cancer is ALL. In some embodiments, the hematological cancer is a dendritic cell tumor. In some embodiments, the hematological cancer is a blast plasmacytoid dendritic cell tumor (BPDCN). In some embodiments, the hematological cancer is B-lineage leukemia.
In any of the foregoing or related aspects and embodiments, the subject in need thereof has a hematological cancer (such as any of the cancers described herein).
In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces a population of FLT3 expressing cells in a subject by at least 60% (e.g., at least 70%, or at least 75%). In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces a population of FLT3 expressing cells in a subject by at least 80% (e.g., at least 90%, at least 95%). The decrease may be a decrease in any one or more of blood, bone marrow cells, and/or cancer cells of the subject relative to baseline.
In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces HSCs and/or HSPCs (e.g., HSCs and early progenitor cells) of a subject by at least 60% (e.g., at least 70%, at least 75%). In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces HSCs and/or HSPCs (e.g., HSCs and early progenitor cells) of a subject by at least 80% (e.g., at least 90%, at least 95%). The decrease may be a decrease in blood and/or bone marrow cells of the subject relative to baseline.
In any of the foregoing or related aspects and embodiments, the administering specifically targets human CD34 + hematopoietic stem cells and/or hematopoietic progenitor cells. In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces cd34+ HSPCs (e.g., HSCs and early progenitor cells) in a subject by at least 60% (e.g., at least 70%, at least 75%). In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces cd34+ HSPCs (e.g., HSCs and early progenitor cells) in a subject by at least 80% (e.g., at least 90%, at least 95%). The decrease may be a decrease in blood and/or bone marrow cells of the subject relative to baseline.
In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces dendritic cells in a subject by at least 60% (e.g., at least 70%, at least 75%). In some embodiments, administration described herein (e.g., in a therapeutically effective amount) reduces dendritic cells in a subject by at least 80% (e.g., at least 90%, at least 95%). The decrease may be a decrease in blood and/or bone marrow cells of the subject relative to baseline.
In some embodiments, the administration reduces the frequency and number of bone marrow lineages in the subject (e.g., reduces the frequency and/or number of bone marrow by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% relative to a baseline level). In some embodiments, administration described herein reduces the circulating myeloid lineage in the subject (e.g., reduces the circulating myeloid lineage by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% relative to baseline levels).
In some embodiments, administration reduces the population of human CD34 +CD38+ cells in the bone marrow mononuclear cells of the subject (e.g., by at least 50%, at least 55%, at least 60%, or at least 65% relative to a baseline level), and/or reduces the population of human CD34 +CD38- cells in the bone marrow mononuclear cells of the subject (e.g., by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% relative to a baseline level).
In some embodiments of the methods described herein, the methods described herein can be effective to treat the cancers described herein (e.g., AML) and/or condition a patient for HSCT. In some embodiments of the methods described herein, the methods described herein are effective to slow the progression of a cancer (e.g., AML) described herein. In some embodiments of the methods described herein, the methods described herein can be effective to reduce tumor burden of a cancer (e.g., AML) described herein. In some embodiments of the methods described herein, the methods described herein are effective to increase survival of a subject having a cancer (e.g., AML) described herein.
In any of the foregoing or related aspects and embodiments, the therapeutically effective amount of an immune cell or population of immune cells expressing an anti-FLT 3 CAR is a dose of about 50,000,000 to 10,000,000,000 cells. In some embodiments, the therapeutically effective amount of an immune cell or population of immune cells expressing an anti-FLT 3 CAR is a dose of about 100,000,000 to 2,000,000,000 cells. In some embodiments, the therapeutically effective amount of an immune cell or population of immune cells expressing an anti-FLT 3 CAR is a dose of about 200,000,000 to 1,000,000,000 cells. In some embodiments, the therapeutically effective amount of an immune cell or population of immune cells expressing an anti-FLT 3 CAR is a dose of about 300,000,000 to 700,000,000 cells.
In any of the foregoing or related aspects and embodiments of the methods described herein, the administering is intravenous. In some embodiments, intravenous administration is by infusion into a subject. In some embodiments, intravenous administration is by bolus injection into the subject.
In some embodiments of the methods described herein, the administering occurs once. In some embodiments of the methods described herein, the administration is once every 3-7 days for 2 to 3 weeks.
In any of the foregoing or related aspects and embodiments of the methods described herein, the method further comprises, prior to the administering step, the steps of:
(i) Collecting blood from a subject;
(ii) Isolation of immune cells (e.g., T cells) from blood;
(iii) Introducing into an isolated immune cell a nucleic acid encoding a CAR of any of the foregoing or related aspects and embodiments; and
(Iv) Amplifying the isolated immune cells obtained in step (iii), wherein the amplification results in immune cells or populations of immune cells administered during the administering step.
In any of the foregoing or related aspects and embodiments, the pharmaceutical compositions described herein further comprise a checkpoint inhibitor. In any of the foregoing or related aspects and embodiments, the methods described herein further comprise administering a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an antagonist of PD1, PD-L1, or CTLA4 (e.g., any such antagonist known in the art, e.g., an antagonistic antibody, such as an antagonistic anti-PD 1 antibody).
In any of the foregoing or related aspects and embodiments, the subject is a human (e.g., a subject treated using any of the methods described herein).
Definition of the definition
As used herein, the term "about" when used to modify a numerical value means that a deviation of up to 10% of the numerical value above and below the value remains within the intended meaning of the value.
As used herein, the term "VL" refers to the light chain variable region of an antibody.
As used herein, the term "VH" refers to the heavy chain variable region of an antibody.
As used herein, the term "percent (%) amino acid sequence identity" or "percent sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence. The percent sequence identity is determined after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity can be accomplished in a variety of ways known in the art. Exemplary alignment tools include, but are not limited to BLASTP, BLAST-2, ALIGN (e.g., ALIGN-2), or Megalign (DNASTAR) software.
Drawings
FIG. 1A shows binding competition of chimeric antibodies 1-18BA (comprising mouse VL (SEQ ID NO: 25) and mouse VH (SEQ ID NO: 27) and human IgG) in REH cells with and without FLT3 ligand.
FIGS. 1B and 1C show the binding affinities of humanized anti-FLT 3 IgG (VL with SEQ ID NO:1 and VH with SEQ ID NO: 3) and humanized anti-FLT 3 scFv (SEQ ID NO:4, C-terminal also comprising a His tag) to REH cells, respectively.
Fig. 2A-2C: fig. 2A is a schematic diagram showing the generation of autologous CAR-T cells and their use as autologous CAR T therapies. Fig. 2B is a schematic diagram showing the CAR structure of an anti-FLT 3 scFV CAR targeting FLT3 expressing cells such as HSPCs, dendritic cells and Acute Myeloid Leukemia (AML). FIG. 2C is a schematic diagram illustrating the mechanism of cell killing of target FLT3 cells by anti-FLT 3CAR T cells; activation of CAR by FLT3 on target cells induces expression of cytokinins and granzymes, thereby inducing apoptosis of the target cells.
Fig. 3A-3D: fig. 3A is an overview of a method for generating anti-FLT 3 CAR T cells and assessing transduction efficiency and cytotoxicity. FIG. 3B is a bar graph showing the change in transduction efficiency of different viral MOIs over time (reported as% GFP + T cells) in T cells transduced with an anti-FLT 3 CAR comprising an anti-FLT 3 scFv encoded by SEQ ID NO:16 (the coding domain is the signal peptide-linker-scFv-CD 8. Alpha. Hinge-CD 8. Alpha. Transmembrane domain-CD 28-41BB-CD3 zeta-T2A-GFP of SEQ ID NO: 4). Figure 3C is a graph showing fold expansion of CAR T cells transduced at MOI 10 (from figure 2A). Figure 3D is a flow chart showing percent (%) GFP expression at different MOIs on day 10 of CAR T cell culture (top) and FLT3 scFv expression using anti-Fab APC antibodies (Jackson ImmunoResearch, numbered 109-607-003) versus GFP expression using CAR T cells of figure 2A (bottom).
Figures 4A-4C show that anti-FLT 3-CAR T cells (as described in figure 3B) are cytotoxic to MOLM-13AML cells. Fig. 4A shows a representative experimental method. FIG. 4B shows a representative flow chart showing the frequency of dead MOLM13 target cells (7-AAD +CellTrace+) at E:T ratios of 1:1 when co-cultured with non-transduced or anti-FLT 3 CAR-T cells for 24 hours (top) and 48 hours (bottom). Figure 4C shows bar graphs representing the mean and standard error of target cell (MOLM 13) killing at the specified E: T ratio at 24 hours (top) and 48 hours (bottom).
Figures 5A-5E show in vivo efficacy of anti-FLT 3 CAR T against MOLM-13AML cells: fig. 5A shows a timeline of implantation of anti-FLT 3 CAR3a T cells (as described in fig. 3B) in mice containing MOLM-13 cells (AML cell line). Fig. 5B is a survival curve of mice treated with control T cells or anti-FLT 3 CAR-T cells for 73 days. Figure 5C shows the overall frequency of human cd45+ MOLM-13 cells in peripheral blood mononuclear fractions before and after treatment with control or CAR-T cells shown for individual mice. Figure 5D shows the frequency of total T cells and gfp+ anti-FLT 3 CAR3a-T cells in peripheral blood mononuclear fraction following treatment with control or gfp+ CAR T cells. FIG. 5E shows the frequency of MOLM-13 cells in the peripheral blood mononuclear fraction following treatment with control T cells or anti-FLT 3 CAR3a-T cells. In fig. 5C and 5D, "X" indicates that no mice were measured in the control group, as all mice had died by day 28.
Figures 6A-6D show successful conditioning using autologous CAR T cells (mice "humanized" by implantation of human pool cells): fig. 6A shows a timeline of mice transplanted with CD123 (cd34+) cells and administered control T cells or anti-FLT 3 CAR T cells (as described in fig. 3B). Figure 6B shows the overall frequency of human CD45 + cells in MNC fractions before and after treatment with control T cells or anti-FLT 3 CAR-T cells shown for individual mice in both cohorts. Fig. 6C shows lineage frequencies (T cells (CD 3 +), B cells (CD 19 +) and myeloid cells (CD 33 +)) before and after treatment with control T cells or anti-FLT 3 CAR-T cells as an average of all mice in both cohorts. Figure 6D shows fold change in lineage frequency versus pre-treatment frequency shown for individual mice in control T cells and anti-FLT 3 CAR T cohorts. Compared to control T cell treated mice, anti-FLT 3 CAR-T cell treated mice showed a significant decrease in myeloid compartments.
Fig. 7A-7D: fig. 7A shows femur and tibia (no gross anatomical differences were observed) of mice transplanted with FLT3-CAR T cells (as described in fig. 3B) and control T cells. Figure 7B shows total cell count and flow cytometry analysis of MNCs from BM (BM-MNCs) and frequency of human cd45+ cells in control T cells and anti-FLT 3-CAR T cell grafts. Fig. 7C shows lineage frequencies (T cells (CD 3 +), B cells (CD 19 +) and myeloid cells (CD 33 +)) in BM-MNC as an average of all mice in both cohorts. Fig. 7D shows lineage cell counts (T cells (CD 3 +), B cells (CD 19 +) and myeloid cells (CD 33 +)) in BM before and after treatment with control or anti-FLT 3 CAR-T cells shown for individual mice in both cohorts.
Fig. 8A-8B: fig. 8A shows a representative contour plot for mcd45-hcd45+lin-gated in control T cells and anti-FLT 3 CAR T (as described in fig. 3B) treated mice (showing significant depletion of hspccd38+cd34+ and cd38-cd34+ populations in anti-FLT 3 CAR T treated mice compared to control) shown for individual mice in control T cells and anti-FLT 3 CAR T treated mice, and a summary plot of cd38+cd34+ and cd38-cd34+hspc is expressed as a percentage of total bone marrow mononuclear cells (BM-MNC). Progenitor cells in bone marrow of anti-FLT 3 CAR T cell treated mice were significantly reduced compared to control mice. FIG. 8B shows the frequency of hematopoietic stem cells (HSC, CD90+CD45RA-) and pluripotent progenitor cells (MPP, CD90-CD45 RA-) as a percentage of total BM-MNC, showing individual mice treated with control T cells or anti-FLT 3 CAR T cells. Progenitor cells in bone marrow of anti-FLT 3 CAR T cell treated mice were significantly reduced compared to control mice.
Fig. 9A-9D: FIG. 9A shows a flow cytometry plot showing the transduction efficiency of suicide CAR vectors measured based on surface expression of anti-FLT 3 scFv in human T cells, showing the frequencies of anti-FLT 3 CAR3a-T cells, anti-FLT 3-CAR3a-EGFRt (the resulting CAR has the amino acid sequence of SEQ ID NO:7 and encodes the domains as signal peptide-linker-CD 8 alpha hinge-CD 8 alpha transmembrane domain-CD 28-41BB-CD3 zeta-T2A-EGFRt) and anti-FLT 3-CAR-iCasp9 (the resulting CAR has the amino acid sequence of SEQ ID NO:8 and encodes the domains as signal peptide-linker-CD 8 alpha hinge-CD 8 alpha transmembrane domain-CD 28-41BB-CD3 zeta-T2A-iCasp 9) cells (35.3%, 27.5% and 16.9%, respectively). FIG. 9B is a schematic diagram of an in vitro cytotoxicity test against target AML NOMO-1 cells (expressing FLT 3) at various effector: target (E: T) cell ratios (10:1, 5:1, 2:1, 1:1, 1:2, and 1:5) for anti-CAR T cells with suicide switch CAR3a-EGFRt or CAR3a-icasp9 compared to original construct CAR3 a. Fig. 9C shows a representative dot plot showing flow cytometry data after debris exclusion after 24 hours of co-culture of effector cells and target cells. Target cells were identified as CellTraceViolet + and effector cells were identified as CellTraceViolet-. The graph shows the frequency of death (7aad+) target cells after gating on CellTraceViolet + cells. FIG. 9D is a bar graph showing the frequency of death (7AAD+) cells at various effector T cell: NOMO-1 target cell ratios for anti-FLT 3 CAR co-cultured with NOMO-1 cells for 24 hours. All FLT3 CAR T cells showed significantly stronger cytotoxic effects on FLT3+ NOMO-1 cells compared to control T cells. There was no significant difference in cytotoxicity between the two suicide CAR T cells and the original CAR construct.
Fig. 10A-10C: fig. 10A shows a flow chart demonstrating surface expression of anti-FLT 3 CAR3a (detection of scFv) and EGFRt (using cetuximab) in T cells transduced with CAR3a-T2A-EGFRt lentiviral vector (as described in fig. 9A). Fig. 10B is a schematic of an in vitro Antibody Dependent Cellular Cytotoxicity (ADCC) assay of CAR3a-T2A-EGFRt T cells. Fig. 10C is a graph showing the percentage (%) of remaining anti-FLT 3 CAR T cells after treatment with different doses of cetuximab, wherein the T cells were cultured alone, with total allogeneic MNC cells, or with T cell depleted allogeneic MNC. Transduced T cells cultured alone showed no significant decrease in CAR3a expressing cells after treatment with cetuximab, whereas transduced cells cultured with total allogeneic MNCs or such MNCs depleted of T cells showed dose-dependent depletion of CAR3a expressing cells by cetuximab. The results support ADCC in vitro against the function of EGFRt-expressing anti-FLT CAR T cells.
Fig. 11A-11D: FIG. 11A shows a timeline of in vivo experiments measuring the survival and frequency of CAR-T cells in peripheral blood of mice containing EGFP-MOLM-13 cells following treatment with CAR3 a-T2A-EGFR-T (as described in FIG. 9A) cells or control T cells. Fig. 11B shows a survival curve generated up to 65 days after AML injection in mice treated with control T cells or anti-FLT 3 CAR3a EFGRt-T cells (with or without cetuximab). FIG. 11C shows the frequency of MOLM-13 (mCD 45-hCD45+EGFP+) cells and T cells (mCD 45-hCD45+CD3+) in Peripheral Blood (PB) at weeks 2, 4 and 6 after treatment with control T cells or anti-FLT 3 CAR3a-T cells (with or without cetuximab). Fig. 11D shows the relative amounts of circulating anti-FLT 3 CAR3a EFGRt-T cells (with and without cetuximab) at weeks 4 and 6 after administration of CAR T cells, as measured by CAR DNA level (normalized to human actin DNA).
Figures 12A-12K show plasmid constructs of CARs. FIG. 12A shows a plasmid expressing the CAR of SEQ ID NO. 16. FIG. 12B shows a plasmid expressing the CAR of SEQ ID NO. 7. FIG. 12C shows a plasmid expressing the CAR of SEQ ID NO. 8. FIG. 12D shows a plasmid expressing the CAR of SEQ ID NO. 6. FIG. 12E shows a plasmid expressing the CAR of SEQ ID NO. 12. FIG. 12F shows a plasmid expressing the CAR of SEQ ID NO. 11. FIG. 12G shows a plasmid expressing the CAR of SEQ ID NO. 10. FIG. 12H shows a plasmid expressing the CAR of SEQ ID NO. 9. FIG. 12I shows a plasmid expressing the CAR of SEQ ID NO. 13. FIG. 12J shows a plasmid expressing the CAR of SEQ ID NO. 14. FIG. 12K shows a plasmid expressing the CAR of SEQ ID NO. 15.
Detailed Description
In certain aspects, and without being limited by any particular mechanism of action, in order to address the challenges of targeting efficacy and resistance in cancer, antibodies, antigen binding fragments, and CAR T cells that can specifically and effectively target and kill Fms-like tyrosine kinase 3 (FLT 3) expressing cells are described herein. The antibodies, fragments, and CAR T cells described herein can target cancer cells (e.g., leukemia cells, such as AML blast cells) as well as FLT3 expressed on the surface of HSCs/HSPCs, and specifically eliminate such cells, allowing for subsequent cancer therapies and/or hematopoietic stem cell transplantation. Antibodies and antigen-binding fragments (e.g., scFv) described herein can bind to extracellular, membrane-proximal FLT3 domains outside of the regions of common mutations in cancer, and do not compete with FLT3 ligands for binding to FLT3. Unlike other known therapies, the antibodies, fragments, CAR T cells, compositions, and methods described herein can target FLT3 expressing cells, regardless of well known mutations in FLT3 receptors.
In one aspect, provided herein are humanized antibodies or antigen-binding fragments thereof (such as heavy chain variable regions (VH), light chain variable regions (VL), and single chain fragments (such as scFV)) that specifically bind FLT3. In certain embodiments, the humanized anti-FLT 3 antibodies and antigen binding fragments thereof provided herein specifically bind to human and monkey (e.g., rhesus monkey) FLT3. In certain embodiments, the humanized anti-FLT 3 antibodies and antigen binding fragments provided herein specifically bind human FLT3.
In another aspect, provided herein are nucleic acids encoding the humanized anti-FLT 3 antibodies and antigen binding fragments provided herein. Also provided herein are vectors comprising nucleic acids encoding the humanized anti-FLT 3 antibodies and antigen binding fragments provided herein. Also provided are cells expressing such nucleic acids to produce such antibodies and fragments, and methods of making such antibodies and fragments.
In one aspect, provided herein are chimeric antibodies that specifically bind FLT3. In certain embodiments, the chimeric anti-FLT 3 antibodies provided herein specifically bind to human and monkey (e.g., rhesus) FLT3. In certain embodiments, the chimeric anti-FLT 3 antibodies provided herein specifically bind to human FLT3. Also provided herein are nucleic acids encoding the chimeric anti-FLT 3 antibodies provided herein. Also provided herein are vectors comprising nucleic acids encoding the chimeric anti-FLT 3 antibodies provided herein. Also provided are cells expressing such nucleic acids to produce such antibodies, and methods of making such antibodies and fragments.
In another aspect, provided herein are recombinant receptors comprising an anti-FLT 3 antibody or antigen binding fragment thereof described herein. In certain embodiments, provided herein are Chimeric Antigen Receptors (CARs) comprising an anti-FLT 3 antibody or antigen binding fragment thereof described herein.
In another aspect, provided herein are immune cells (e.g., CAR T cells) comprising a CAR described herein.
In yet another aspect, provided herein are methods of using the humanized anti-FLT 3 antibodies described herein, antigen binding fragments thereof, recombinant receptors (such as CARs), and immune cells (e.g., CAR T cells). In certain embodiments, provided herein are methods of treating hematological malignancies (e.g., AML) using anti-FLT 3CAR immune cells (e.g., by administering anti-FLT 3CAR T cells to a human). In certain embodiments, provided herein are methods of HSC transplantation conditioning using anti-FLT 3CAR T cells (e.g., by administering anti-FLT 3CAR T cells to a human). In some embodiments, the HSC transplantation conditioning method may be followed by hematopoietic cell transplantation. In certain embodiments, provided herein are methods of treating hematological malignancies (e.g., AML) using an anti-FLT 3 antibody or antigen binding fragment thereof (e.g., by administering an anti-FLT 3 antibody or fragment to a human). In certain embodiments, provided herein are methods of HSC transplant conditioning using an anti-FLT 3 antibody or antigen binding fragment thereof (e.g., by administering an anti-FLT 3 antibody or fragment to a human). In some embodiments, the HSC transplantation conditioning method may be followed by hematopoietic cell transplantation.
Anti-FLT 3 antibodies
Provided herein are antibodies and antigen-binding fragments thereof that bind FLT 3. Reference herein to an antibody fragment refers to an antigen-binding fragment of the described antibodies. In certain embodiments, provided herein are antibodies and fragments thereof that specifically bind to human and rhesus FLT 3. In certain embodiments, provided herein are antibodies and fragments thereof that specifically bind to human FLT 3. The antibodies and fragments described herein may exhibit cross-reactivity with FLT3 from one or more other species (other than human and rhesus). In some embodiments, antibodies and fragments thereof that specifically bind to human and/or monkey (e.g., rhesus) FLT3 and that do not exhibit cross-reactivity with FLT3 from other species are also contemplated. In certain embodiments, provided herein are humanized antibodies and antigen-binding fragments thereof that bind FLT 3. In certain embodiments, provided herein are chimeric antibodies and antigen-binding fragments thereof that bind FLT 3.
In some embodiments, contemplated anti-FLT 3 antibodies and fragments comprise any of the CDRs described herein. In some embodiments, provided herein is a single-chain variable fragment (scFV) comprising any of the CDRs described herein. In some embodiments, contemplated anti-FLT 3 antibodies and fragments comprise any of the light chain variable regions described herein and/or any of the heavy chain variable regions described herein. In some embodiments, provided herein is a single chain variable fragment (scFV) comprising any of the light chain variable regions described herein and/or any of the heavy chain variable regions described herein.
In some embodiments, contemplated humanized anti-FLT 3 antibodies and fragments comprise any of the CDRs described herein. In some embodiments, contemplated humanized anti-FLT 3 antibodies and fragments comprise any of the light chain variable regions described herein and/or any of the heavy chain variable regions described herein.
In some embodiments, the anti-FLT 3 antibodies and fragments comprise a light chain variable region having a sequence at least 95% identical to any of the light chain variable regions described herein and/or a heavy chain variable region having a sequence at least 95% identical to any of the heavy chain variable regions described herein. In some embodiments, provided herein are single chain variable fragments (scFv) comprising a sequence having at least 95% identity to any of the light chain variable regions described herein and/or heavy chain variable regions comprising a sequence having at least 95% identity to any of the heavy chain variable regions described herein.
In some embodiments, the anti-FLT 3 antibodies and fragments comprise a light chain variable region having a sequence with at least 95% identity (at least 97% identity in the CDR regions) to any light chain variable region described herein and/or a heavy chain variable region having a sequence with at least 95% identity (at least 97% identity in the CDR regions) to any heavy chain variable region described herein. In some embodiments, provided herein are single chain variable fragments (scFV) comprising a sequence having at least 95% identity (at least 97% identity in CDR regions) to any light chain variable region described herein and/or a heavy chain variable region having a sequence having at least 95% identity (at least 97% identity in CDR regions) to any heavy chain variable region described herein.
Complementarity determining regions
Complementarity Determining Regions (CDRs) are defined in a variety of ways in the art, including Kabat, chothia, abM, contact and IMGT. In some embodiments, the CDRs of an antibody are defined according to the Kabat system. The Kabat system is based on sequence variability (see, e.g., kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, 5 th edition, U.S. Pat. No. HEALTH AND Human Services, NIH publication No. 91-3242;Kabat EA&Wu TT (1971) ANN NY ACAD SCI 190:382-391). In some embodiments, the CDRs of the antibodies described herein are determined using the Kabat system.
In some embodiments, the CDRs of an antibody are defined according to the Chothia system. Choth ia systems are based on the position of immunoglobulin structural loop regions (see, e.g., tramontano A et al, (1990) J Mol Biol 215 (1): 175-82;Chothia C&Lesk AM, (1987) J Mol Biol 196:901-917; U.S. Pat. No. 7,709,226; al-Lazik ani B et al, (1997) J Mol Biol 273:927-948; and Chothia C et al, (1992) J Mol Biol 227:799-817). The term "Chothia CDR" and similar terms are art-recognized and refer to antibody CDR sequences determined according to the methods of Chothia and Lesk,1987, j.mol. Biol.,196:901-917 (see also, e.g., U.S. patent nos. 7,709,226 and Martin,A.,"Protein Sequence and Structure Analysis of Antibody Variable Domains,"in Antibody Engineering,Kontermann and Diibel, editions, chapter 31, pages 422-439, springer-Verlag, berlin (2001)). In some embodiments, the CDRs of the antibodies described herein are determined using the Chothia system.
In some embodiments, the CDRs of an antibody are defined according to the AbM system. The AbM system is based on hypervariable regions representing a tradeoff between Kabat CDRs and Chothia structural loops, and wherein the CDRs are determined using Oxford Molecular AbM antibody modeling software (Oxford Molecular Group, inc.). In some embodiments, the CDRs of the antibodies described herein are determined using the AbM numbering system.
In some embodiments, the CDRs of an antibody are defined according to the IMGT system (see'International ImMunoGeneTicsWebsite imgt. Org, creator and director: marie-Paule Lefranc, montpellier, france; see, e.g., lefranc, m. -p.et al, 1999,Nucleic Acids Res, 27:209-212 and Lefranc, m. -p. 1999,The Immunologist,7:132-136 and Lefranc, m. -p.et al, 1999,Nucleic Acids Res, 27:209-212). In some embodiments, the CDRs of the antibodies described herein are determined using an IMGT system.
In some embodiments, the CDRs of an antibody are defined according to the Contact system. The Contact definition is based on analysis of available complex crystal structures (bioif. Org. Uk/abs) (see, e.g., ,Martin A."Protein Sequence and Structure Analysis of Antibody Variable Domains,"in Antibody Engineering,Kontermann and Diibel editions, chapter 31, pages 422-439, springer-Verlag, berlin (2001) and MacCallum RM et al, (1996) J Mol Biol 5:732-745). In some embodiments, the CDRs of the antibodies described herein are determined using a Contact system.
Kabat, chothia, abM, IMGT and/or Contact CDR positions may vary depending on the antibody and may be determined according to methods known in the art.
In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having a light chain variable region comprising complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID No. 86. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof having a light chain variable region comprising complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID No. 87. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having a light chain variable region comprising complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO 88. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 having SEQ ID NOS 86, 87 and 88, respectively. In certain embodiments, the anti-FLT 3 antibody or fragment is humanized.
In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having a heavy chain variable region comprising complementarity determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID No. 89. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having a heavy chain variable region comprising complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID No. 90. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having a heavy chain variable region comprising complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO. 91. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof having heavy chain variable regions comprising CDR-H1, CDR-H2 and CDR-H3 having SEQ ID NOS 89, 90 and 91, respectively. In certain embodiments, the anti-FLT 3 antibody or fragment is humanized.
In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a light chain variable region comprising CDR-L1 of SEQ ID NO:86, CDR-L2 of SEQ ID NO:87, and/or CDR-L3 of SEQ ID NO:88, and/or (ii) a heavy chain variable region comprising CDR-H1 of SEQ ID NO:89, CDR-H2 of SEQ ID NO:90, and/or CDR-L3 of SEQ ID NO: 91. In certain embodiments, the anti-FLT 3 antibody or fragment is humanized.
In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a light chain variable region comprising CDR-L1 of SEQ ID NO:86, CDR-L2 of SEQ ID NO:87, and CDR-L3 of SEQ ID NO:88, and (ii) a heavy chain variable region comprising CDR-H1 of SEQ ID NO:89, CDR-H2 of SEQ ID NO:90, and CDR-L3 of SEQ ID NO: 91. In certain embodiments, the anti-FLT 3 antibody or fragment is humanized.
In certain embodiments, the CDRs of the antibodies described herein are determined using the Kabat system.
In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising the CDRs of any of the antibodies described herein, which are defined according to any of the CDR definition systems described above (e.g., kabat). In certain embodiments, the anti-FLT 3 antibody or fragment is humanized.
In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) one, two, or all three CDRs of the variable region of SEQ ID NO:28, and/or (ii) one, two, or all three CDRs of the variable region of SEQ ID NO: 17. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising three CDRs of the variable region of SEQ ID NO:28 and three CDRs of the variable region of SEQ ID NO: 17. In certain embodiments, the anti-FLT 3 antibody or fragment is humanized (e.g., a humanized antibody or fragment of an anti-FLT 3 antibody having a heavy chain variable region comprising SEQ ID NO:17 and/or a light chain variable region comprising SEQ ID NO: 28). In specific embodiments, the CDRs are determined by Kabat.
In some embodiments, provided herein are anti-FLT 3 antibodies, or fragments thereof (e.g., scFv), comprising one, two, three, four, five, or all six CDRs of any of the mouse anti-FLT 3 antibodies described in U.S. patent publication No. 20190127464. In some embodiments, provided herein are anti-FLT 3 antibodies, or fragments thereof (e.g., scFv), comprising one, two, three, four, five, or all six CDRs of a mouse anti-FLT 3 antibody described in U.S. patent publication No. 20190389955, which has a VL of SEQ ID No. 25 and a VH of SEQ ID No. 27 (based on the SEQ ID NO in U.S. patent publication No. 20190389955). In some embodiments, provided herein are anti-FLT 3 antibodies, or fragments thereof (e.g., scFv), comprising all six CDRs of any mouse anti-FLT 3 antibody described in U.S. patent publication No. 20190127464 (e.g., the antibody described in U.S. patent publication No. 20190127464a, with VL of SEQ ID NO:5 and VH of SEQ ID NO: 7). In certain embodiments, the anti-FLT 3 antibody or fragment is humanized. In specific embodiments, the CDRs are determined by Kabat.
Also contemplated herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) having substitutions, deletions or insertions in the CDR sequences described above. In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) that have at least 97% CDR sequence identity to the CDRs described herein. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) that have at least 98% CDR sequence identity to the CDRs described herein. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) that have at least 99% CDR sequence identity to the CDRs described herein. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) having one, two, or up to three substitutions, deletions, or insertions in the CDR sequences described herein. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) having one, two, or at most two substitutions, deletions, or insertions in any of the CDR sequences described herein. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) having one, two, three, four, five, six, seven, eight, nine, or up to ten substitutions, deletions, or insertions in total in the six CDRs of the antibodies and fragments described herein. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) having a total of one, two, or up to three substitutions, deletions, or insertions in the six CDRs of the antibodies and fragments described herein. In certain embodiments, the anti-FLT 3 antibody or fragment is humanized.
As known in the art, CDRs are surrounded by framework regions. In certain embodiments, an anti-FLT 3 antibody or fragment described herein has a human framework region or a human derived framework region. In some embodiments of the anti-FLT 3 antibodies and fragments described herein, the framework regions are human framework regions. In some embodiments of the anti-FLT 3 antibodies and fragments described herein, the framework regions are human-derived framework regions.
Usable and known in the art human framework regions include, but are not limited to: (i) a human germline framework region, (ii) a human mature (somatic mutation) framework region, (iii) a framework region selected using a "best fit" method, (iv) a framework region derived from the consensus sequences of human antibodies of a particular subset of the light and heavy chain variable regions, and (v) a framework region derived from a screened FR library. See, e.g., baca et al, J.biol. Chem.272:10678-10684 (1997); chothia et al, J.mol.biol.278:457-479 (1998); carter et al Proc.Natl. Acad. Sci.USA,89:4285 (1992); presta et al J.Immunol.,151:2623 (1993); sims et al J.Immunol.151:2296 (1993); rosok et al, J.biol. Chem. M.271:22611-22618 (1996); and Almagro and Fransson, front. Biosc i.13:1619-1633 (2008).
Any of the CDRs described herein can be inserted into any of the framework regions described herein using known DNA recombination techniques.
Exemplary anti-FLT 3 antibodies: VL and VH
In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a heavy chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs 3 and 17-27. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 3 and 17-27. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 3 and 17-27. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from any one of SEQ ID NOs 3 and 17-27, at least 95% (or at least 96%, 97%, 98%, 99%, or 100%) identity in the framework region, and at least 97% (or at least 98%, 99%, or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a light chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs 1,2 and 28-38. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a light chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 1,2 and 28-38. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a light chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 1,2 and 28-38. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising a light chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from any one of SEQ ID NOs 1,2, and 28-38, at least 95% (or at least 96%, 97%, 98%, 99%, or 100%) identity in the framework region, and at least 97% (or at least 98%, 99%, or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a heavy chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs 3 and 17-27, and (ii) a light chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs 1,2 and 28-38. In some embodiments, provided herein are anti-FLT 3 antibodies, or fragments thereof (e.g., scFv), comprising (i) a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from any one of SEQ ID nos. 3 and 17-27, and (ii) a light chain variable region comprising an amino acid sequence having at least 1%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID nos. 1 2 and 28-38 has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, provided herein are anti-FLT 3 antibodies, or fragments thereof (e.g., scFv), comprising (i) a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 3 and 17-27, and (ii) a light chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 1, 2, and 28-38, amino acid sequence of at least 98%, or at least 99% identity. in certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). in certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a heavy chain variable region comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from any of SEQ ID NOs 3 and 17-27, having at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and having at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and (ii) a light chain variable region, Comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence selected from any of SEQ ID NOs 1, 2 and 28-38, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In some embodiments, contemplated herein is an anti-FLT 3 antibody or fragment thereof (e.g., scFv) comprising any of the light chain variable regions and any of the heavy chain variable regions described above.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:3, and/or (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:3, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:3, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:1, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:3, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:3, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:3, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:2, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:18, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:18, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 29. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:18, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:29, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:19, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:19, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 30. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:19, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:30, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:20, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:20, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:20, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:31, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID No. 21, and/or (ii) a VL comprising the amino acid sequence of SEQ ID No. 32. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:21, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:21, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:32, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID No. 22, and/or (ii) a VL comprising the amino acid sequence of SEQ ID No. 33. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:22, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:22, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:33, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:23, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:23, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 34. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:23, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:34, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID No. 24, and/or (ii) a VL comprising the amino acid sequence of SEQ ID No. 35. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:24, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 35. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:24, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:35, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:25, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:25, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 36. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:25, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:36, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID No. 26, and/or (ii) a VL comprising the amino acid sequence of SEQ ID No. 37. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:26, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 37. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:26, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:37, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:27, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:27, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 38. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:27, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:38, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are humanized anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
In certain embodiments, provided herein are chimeric anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:17, and/or (ii) a VL comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:17, and/or (ii) a VL comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 28. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising (i) a VH comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:17, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region, and/or (ii) a VL comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:28, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region. In some embodiments, provided herein are chimeric anti-FLT 3 antibodies or fragments thereof (e.g., scFv) comprising both VH and VL comprising the sequences specified in this paragraph.
scFv
In certain embodiments, provided herein are scFv fragments of the humanized anti-FLT 3 antibodies described herein. In certain embodiments, provided herein are scFv fragments comprising any VH and/or VL described herein, including any VH and VL pair described herein. Methods for preparing single chain variable fragment antibodies are known in the art. For example, scFv antibodies can be prepared by fusing a heavy chain variable region (VH) to a light chain variable region via a short peptide linker. Suitable short peptide linkers are known in the art, and exemplary linkers are described herein.
In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence selected from any one of SEQ ID NOs 4, 5 and 40-49. In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs 4, 5, and 40-49. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with an amino acid sequence selected from any one of SEQ ID NOs 4, 5 and 40-49, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence selected from any one of SEQ ID NOs 4,5, 44-47 and 49. In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs 4,5, 44-47 and 49. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with an amino acid sequence selected from any one of SEQ ID NOs 4,5, 44-47 and 49, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising the amino acid sequence of SEQ ID No. 4. In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 4. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 4, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising the amino acid sequence of SEQ ID No. 5. In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 5. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3 scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 5, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising the amino acid sequence of SEQ ID NO. 44. In some embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 44. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 44, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising the amino acid sequence of SEQ ID NO. 45. In some embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 45. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 45, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising the amino acid sequence of SEQ ID NO. 46. In some embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 46. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 46, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising the amino acid sequence of SEQ ID NO. 47. In some embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 47. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 47, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising the amino acid sequence of SEQ ID No. 49. In some embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 49. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID No. 49, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 39. In certain embodiments, substitutions, insertions, or deletions in these sequences occur in regions outside the CDRs (i.e., in the framework regions). In certain embodiments, provided herein are anti-FLT 3scFv fragments comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO:39, at least 95% (or at least 96%, 97%, 98%, 99% or 100%) identity in the framework region, and at least 97% (or at least 98%, 99% or 100%) identity in the CDR region.
Linkers useful for scFv
In some embodiments, the disclosure provides an anti-FLT 3 single chain variable fragment (scFv) comprising one or more linkers connecting a VH and a VL. A "linker" is a functional group that covalently links two or more polypeptides or nucleic acids to each other. The linker may be any linker known in the art. In some embodiments, the linker comprises a hydrophilic amino acid. In some embodiments, the linker comprises glycine and serine.
In some embodiments, the linker is of the formula (Gly 3-4-Ser)1-4. In some embodiments, the linker is a Gly 4 Ser linker that is repeated 1 to 4 times, in some embodiments, the linker is a Gly 3 Ser linker that is repeated 1 to 4 times, in some embodiments, the linker comprises Gly 4 Ser and Gly 3 Ser linkers that are each repeated 1 to 4 times.
In certain embodiments, the linker is 4 to 25 amino acids in length. In certain embodiments, the linker is 4 to 21 amino acids in length. In some embodiments, the linker is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 20 amino acids in length.
In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 50. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 51. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 52. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 53. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 54.
In some embodiments, the linker comprises the nucleotide sequence of SEQ ID NO. 55. In some embodiments, the linker comprises the nucleotide sequence of SEQ ID NO. 56. In some embodiments, the linker comprises the nucleotide sequence of SEQ ID NO. 57. In some embodiments, the linker comprises the nucleotide sequence of SEQ ID NO. 58. In some embodiments, the linker comprises the nucleotide sequence of SEQ ID NO. 59.
In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising any linker that connects any light chain variable region (VL) described herein to any heavy chain variable region (VH) (or any VL/VH pair described herein). In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising any of the linkers described herein connecting a VL (comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID NO: 38) to a VH (comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID NO: 27).
In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising a linker of SEQ ID NO:50 connecting VL (which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID NO: 38) to VH (which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID NO: 27).
In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising a linker of SEQ ID NO:51 connecting VL (comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID NO: 38) to VH (comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID NO: 27).
In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising a linker of SEQ ID No. 52 connecting a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID No. 38 to a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID No. 27.
In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising a linker of SEQ ID NO:53 connecting VL (which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID NO: 38) to VH (which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID NO: 27).
In some embodiments, provided herein are anti-FLT 3 scFv fragments comprising a linker of SEQ ID No. 54 connecting VL (comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID No. 38) to VH (comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID No. 27).
Other anti-FLT 3 antibodies, fragments and Properties
In some embodiments, described herein are anti-FLT 3 antibodies, wherein the antibodies are immunoglobulins comprising any of the VH and VL regions described herein. The immunoglobulin molecules that can be used are of any type (e.g., igG, igE, igM, igD, igY, igA). Immunoglobulin molecules that can be used are of any class (e.g., igG1, igG2, igG3, igG4, igA1, igA 2). Immunoglobulin molecules that can be used are of any subclass. In some embodiments, the immunoglobulin is IgG.
In some embodiments, described herein are single domain anti-FLT 3 antibodies having only heavy chains or only light chains (including any VH or VL described herein). In some embodiments, described herein are single domain anti-FLT 3 antibodies having only heavy chains (including any VH described herein).
In some embodiments, described herein are antigen binding fragments of anti-FLT 3 antibodies, including but not limited to Fv fragments, fab fragments, F (ab') 2 fragments, or disulfide-linked Fv (sdFv).
In some embodiments, described herein are chimeric anti-FLT 3 antibodies, or antigen binding fragments thereof, wherein the chimeric antibodies have a murine variable region and a constant region of another species (e.g., human).
In some embodiments, described herein are multispecific anti-FLT 3 antibodies and fragments (e.g., bispecific antibodies and fragments) that specifically bind to one or more additional antigens (e.g., a second additional antigen) in addition to FLT3 (using the antigen binding fragments described herein). One or several additional antigens may be antigens that are exposed on the surface of target cells (e.g., AML cells).
In some embodiments, described herein are anti-FLT 3 antibodies and fragments thereof that have a binding affinity for FLT3 protein of EC50 of about 0.1nM to 100nM, 0.5nM to 50nM, or 1nM to 10 nM. In some embodiments, described herein are anti-FLT 3 antibodies and fragments thereof that have a binding affinity for FLT3 protein of an EC50 of less than about 100nM, less than about 75nM, less than about 50nM, less than about 25nM, less than about 10nM, less than about 5nM, less than about 3nM, less than about 2nM, or less than about 1 nM. In some embodiments, described herein are anti-FLT 3 antibodies and fragments thereof that have a binding affinity for FLT3 protein of an EC50 of less than about 15nM, less than about 10nM, less than about 5nM, or less than about 2.5 nM.
In some embodiments, described herein are anti-FLT 3 antibodies and fragments thereof that mediate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is triggered when an antibody that binds to the cell surface interacts with an Fc receptor on a Natural Killer (NK) cell, as known in the art; NK cells express the receptor Fc. γ.RIII (CD 16), which recognizes the IgG1 and IgG3 subclasses. ADCC killing mechanisms involve the release of cytoplasmic granules containing perforin and granzyme.
In some embodiments, the anti-FLT 3 antibodies and fragments thereof (e.g., scFv) described herein do not compete with FLT3 ligands for binding to FLT3. In some embodiments, the binding of anti-FLT 3 antibodies and fragments described herein to FLT3 in the presence of the FLT3 ligand (or after pretreatment of the cells with the FLT3 ligand) is substantially the same as in the absence of the FLT3 ligand (or without pretreatment of the cells with the FLT3 ligand) (e.g., in vitro) (using any method known in the art or described herein to assess competitive binding, see e.g., example 1). In some embodiments, the anti-FLT 3 antibodies and fragments described herein reduce binding to FLT3 by less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, or less than 1% in the presence of FLT3 ligand (or after pretreatment of cells with FLT3 ligand) relative to the absence of FLT3 ligand (or without pretreatment of cells with FLT3 ligand) (e.g., in vitro) (using any methods known in the art or described herein to assess competitive binding, see, e.g., example 1). In some embodiments, the anti-FLT 3 antibodies and fragments described herein reduce binding to FLT3 by less than 5%, less than 3%, or less than 1% in the presence of FLT3 ligand (or after pretreatment of cells with FLT3 ligand) relative to the absence of FLT3 ligand (or without pretreatment of cells with FLT3 ligand) (e.g., in vitro) (using any methods known in the art or described herein to assess competitive binding, see e.g., example 1).
Provided herein are anti-FLT 3 antibodies described herein, and fragments thereof (e.g., scFv), which can target and eliminate or kill FLT3 expressing cells. Provided herein are anti-FLT 3 antibodies and fragments thereof (e.g., scFv) that can target FLT3 expressed on the surface of cells. For example, provided herein are anti-FLT 3 antibodies and fragments thereof described herein that can target FLT3 expressed on the surface of cancer cells (e.g., leukemia cells, such as AML blasts). Also provided herein are anti-FLT 3 antibodies described herein, and fragments thereof, that can target FLT3 expressed on HSCs and/or HSPC surfaces. Also provided herein are anti-FLT 3 antibodies described herein, and fragments thereof, that can target FLT3 expressed on the surface of any hematopoietic cell lineage described herein or known in the art that expresses FLT3. Without being bound by any theory or mechanism of action, the anti-FLT 3 antibodies and fragments thereof (e.g., scFv) described herein can bind to the extracellular, membrane-proximal FLT3 domain.
In some embodiments, the anti-FLT 3 antibodies and fragments thereof described herein bind wild-type and mutant FLT3 (e.g., FLT3 that is known or determined to be mutated in cancer, such as cancer treated with such antibodies/fragments). In some embodiments, the anti-FLT 3 antibodies and fragments thereof described herein bind to a FLT3 region that is not mutated in cancer (e.g., is not known to be mutated in cancer (such as cancer treated with the antibodies/fragments) or is determined to be not mutated in cancer). In some embodiments, the anti-FLT 3 antibodies and fragments thereof described herein bind to or target (e.g., kill) FLT3 expressing cells, whether the cells express wild-type or mutant FLT3. In some embodiments, the anti-FLT 3 antibodies and fragments thereof described herein bind to or target (e.g., kill) FLT3 expressing cancer cells that express wild-type and mutant FLT3. In some embodiments, the anti-FLT 3 antibodies and fragments thereof described herein bind to or target (e.g., kill) FLT3 expressing mutant FLT3 cancer cells (e.g., known to express mutant FLT3 or determined to express mutant FLT3, such as with any mutation in FLT3 known in the art).
Preparation of antibodies
The anti-FLT 3 antibodies and antigen binding fragments thereof described herein may be prepared by any method known in the art and/or described herein.
Methods for preparing monoclonal antibodies are known in the art, for example using hybridoma technology. See, e.g., harlow E and Lane D, antibodies: A Laboratory Manual (Cold Spring Harbor Press, 2 nd edition 1988); HAMMERLING GJ et al, in: monoclonal Antibodies and T-Cell Hybridomas 563 (Elsevier, N.Y., 1981); kohler G and MILSTEIN C,1975,Nature 256:495; goding JW (eds.), monocolonal Antibodies: PRINCIPLES AND PRACTICE, pages 59-103 (ACADEMIC PRESS, 1986). In using hybridoma technology, a mouse or another suitable host animal may be immunized with a target protein (e.g., FLT 3) to elicit lymphocytes that produce antibodies that will specifically bind to the target protein, and then the lymphocytes are fused with myeloma cells to form the hybridoma. The hybridoma cells were then grown in culture and assayed for antibody production. The binding specificity of antibodies produced by this method can be determined by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or Radioimmunoassay (RIA). The monoclonal antibodies may be further purified.
Monoclonal antibodies can also be prepared using recombinant and phage display techniques and using humanized mice. See, e.g., brinkman U et al, 1995,J.Immunol.Methods 182:41-50; ames RS et al 1995,J Immunol.Methods 184:177-186; laffleur et al, 2012,Methods Mol.Biol.901:149-59; persic L. et al, 1997, gene 187:9-18.
Methods of making chimeric antibodies are known in the art. See, e.g., morrison SL,1985,Science 229:1202-7; GILLIES SD et al, 1989,J.Immunol.Methods 125:191-202; oi VT & Morrison SL,1986,BioTechniques 4:214-221. When chimeric antibodies are produced, the variable region of one species (e.g., murine) is linked to the constant region of another species (e.g., human).
Methods of making humanized antibodies are known in the art, including but not limited to grafting by CDR. See, e.g., padlan EA (1991) Mol Immunol 28 (4/5): 489-498; studnicka GM et al, (1994) Prot Engineering 7 (6): 805-814; and Roguska MA et al, (1994) PNAS 91:969-973; tan P et al, (2002) J Immunol 169:1119-25; caldas C et al, (2000) Protein Eng.13 (5): 353-60; morea V et al, (2000) Methods 20 (3): 267-79; baca M et al, (1997) J Biol Chem 272 (16): 10678-84; roguska MA et al, (1996) Protein Eng 9 (10): 895904; couto JR et al, (1995) Cancer Res.55 (23 support): 5973s-5977s; couto JR et al, (1995) CANCER RES (8): 1717-22; sandhu JS (1994) Gene 150 (2): 409-10; pedersen JT et al, (1994) J Mol Biol 235 (3): 959-73).
Methods of making human antibodies are known in the art and include phage display methods using libraries of antibodies derived from human immunoglobulin sequences. See, for example, international publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.
Methods for preparing antibody fragments, including single chain Fv (scFv), are also known in the art. See, for example, ahmad et al 2012,Clinical and Developmental Immunol ogy,doi:10.1155/2012/980250; wang et al, 2006, anal. Chem.78,997-1004; pansri et al, 2009,BMC Biotechnology 9:6. For example, scFv can be constructed by fusing the heavy and light chain variable regions via a short polypeptide linker (using recombinant expression techniques), and scFv antibodies with desired antigen binding properties can be selected by methods known in the art. In addition, fab and F (ab') 2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules using papain and pepsin, respectively.
Methods of making single domain antibodies (e.g., without light chains) are also known in the art. See, e.g., RIECHMANN L & Muyldermans S,1999,J Immunol.231:25-38; nuttall SD et al 2000,Curr Pharm Biotechnol.1 (3): 253-263; muyldermans S,2001,J Biotecnol 74 (4): 277-302.
Methods for preparing bispecific antibodies are well known in the art. See, e.g., konterman,2012, MAbs 4:182-197; gramer et al, 2013, MAbs 5:962-973.
Methods of making mouse anti-FLT 3 antibodies are described in U.S. patent publication No. 20190137464 and U.S. patent publication No. 20190389955, each of which is incorporated herein by reference in its entirety, and specifically describe the preparation of mouse anti-FLT 3 antibodies. Methods of making humanized anti-FLT 3 antibodies and chimeric anti-FLT 3 antibodies are described herein (see, e.g., examples).
Methods for recombinantly producing antibodies are also known in the art. In some embodiments, for recombinant production of an anti-FLT 3 antibody (or antigen binding fragment thereof), a nucleic acid encoding the antibody (or antigen binding fragment thereof) is isolated and inserted into one or more vectors for expression in a host cell. In some embodiments, a method of producing an anti-FLT 3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and recovering the antibody from the host cell (or host cell culture medium), and optionally further purifying the antibody. In some embodiments, a method of making an antigen-binding fragment of an anti-FLT 3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the fragment under conditions suitable for expression of the fragment, and recovering the fragment from the host cell (or host cell culture medium), and optionally further purifying the fragment.
Recombinant receptors, such as chimeric antigen receptors
In one aspect, provided herein is a recombinant receptor comprising any of the anti-FLT 3 antibodies or antigen binding fragments thereof described herein. In some embodiments, provided herein are recombinant receptors comprising any antigen-binding fragment of any of the anti-FLT 3 antibodies described herein. In some embodiments, provided herein are recombinant receptors comprising any anti-FLT 3 VH and/or VL described herein. In some embodiments, provided herein are recombinant receptors comprising any of the anti-FLT 3 scFv described herein. Contemplated recombinant receptors include functional non-TCR antigen receptors. In some embodiments, provided herein are chimeras of a signaling domain of a T Cell Receptor (TCR) complex and a FLT3 antigen recognition domain (e.g., an anti-FLT 3 scFv, such as any of those described herein). In some embodiments, the recombinant receptor provided herein is a Chimeric Antigen Receptor (CAR). Also provided herein are cells (e.g., immune cells) that express a recombinant receptor (e.g., CAR) described herein. The CAR-expressing T cells are referred to herein as CAR T cells. Also provided herein is a use of a cell (e.g., an immune cell) expressing a recombinant receptor (e.g., CAR) described herein in therapy, such as treatment of a disease associated with FLT3 expression. In some embodiments, provided herein is the use of a cell (e.g., an immune cell) expressing a recombinant receptor (e.g., CAR) described herein in the treatment of cancer (e.g., AML, ALL, or dendritic cell tumor). In some embodiments, provided herein is the use of cells (e.g., immune cells) expressing a recombinant receptor (e.g., CAR) described herein to condition a subject prior to hematopoietic cell transplantation.
Examples of antigen receptors (including CARs) are well known in the art. Methods for their preparation are also well known in the art. See, e.g., sadelain et al, cancer discover.2013 april;3 (4) 388-398; davila et al, 2013, PLOS ONE 8 (4): e61338; turtle et al, curr.Opin.Immunol.,2012,24 (5): 633-39; wu et al, cancer,2012,18 (2): 160-75.
The CARs provided herein generally comprise an extracellular domain comprising any anti-FLT 3 antibody or fragment described herein (e.g., any anti-FLT 3 antigen binding fragment described herein). In certain embodiments, a CAR provided herein further comprises a transmembrane domain (such as any of the transmembrane domains described herein) and an intracellular domain (such as any of the intracellular domains described herein). In some embodiments, the CARs provided herein further comprise a linker between the extracellular domain and the transmembrane domain and/or between the transmembrane domain and the intracellular domain. Described herein are exemplary linkers that can be used in the CARs provided herein. In some embodiments, the linker comprises a hydrophilic amino acid. In some embodiments, the linker comprises glycine and serine.
Four-generation Chimeric Antigen Receptors (CARs) are known in the art. The first generation CARs linked antibody-derived scFv to the CD3zeta (ζ or z) intracellular signaling domain of T cell receptors via a hinge and a transmembrane domain. The second generation CARs incorporate additional domains into the intracellular signaling domain (e.g., CD28, 4-1BB (41 BB) or ICOS) to provide a costimulatory signal. Third generation CARs contain two costimulatory domains (fused to the TCR cd3ζ chain). The third generation costimulatory domain may comprise, for example, any combination of at least two of CD27, CD28, 4-1BB, ICOS and 0X 40. The fourth generation CAR may comprise one or more stimulatory cytokines. Examples of CARs include CARs comprising: an extracellular antigen binding domain (e.g., comprising an antigen binding scFv), a linker or hinge region, a transmembrane domain, and an intracellular domain comprising one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3z and/or a co-stimulatory molecule (Maude et al, blood.2015;125 (26): 4017-4023; kakarla and Gottschalk, cancer J.2014;20 (2): 151-1 55). Functionally, the CD3z signaling domain of the T cell receptor will activate and induce T cell proliferation when bound, but may lead to anergy (lack of response of the body defense mechanisms leading to direct induction of peripheral lymphocyte tolerance). Lymphocytes are considered unresponsive when they are unable to respond to a particular antigen. The addition of a costimulatory domain in the second generation CAR may increase the replication capacity and persistence of the modified T cells. Third generation CARs combine multiple signaling domains (co-stimulatory) that can enhance potency. Fourth generation CAR expression can improve expansion and persistence of stimulatory cytokines after implantation. Provided herein are any such CAR, wherein the extracellular domain comprises an anti-FLT 3 antigen binding fragment (e.g., any anti-FLT 3 antigen binding fragment described herein, e.g., scFv).
In some embodiments, provided herein are first generation CARs. In some embodiments, provided herein are second generation CARs. In some embodiments, third generation CARs are provided herein. In some embodiments, provided herein are fourth generation CARs.
In some embodiments, the CAR comprises: an extracellular (ecto) domain comprising an anti-FLT 3 antigen binding domain (e.g., scFv); a transmembrane domain; and an intracellular (intracellular) domain. In some embodiments, the CAR comprises: an extracellular (ecto) domain comprising an anti-FLT 3 antigen binding domain (e.g., scFv); a transmembrane domain; and an intracellular domain comprising an activation domain and a co-stimulatory domain.
Extracellular domain/ectodomain
In certain embodiments, the extracellular domain comprises any of the anti-FLT 3 antibodies or antigen binding fragments thereof described herein (see, e.g., the disclosure in the "anti-FLT 3 antibody" section of anti-FLT 3 antibodies and fragments thereof contemplated hereinabove, including sub-sections describing anti-FLT 3 VH and/or VL regions that may be used). In certain embodiments, the extracellular domain comprises any of the anti-FLT 3 single chain variable fragments (scFv) described herein (see, e.g., the disclosure in the "anti-FLT 3 antibody" section above, including sub-sections describing anti-FLT 3 scFv, VH and/or VL regions useful in scFv, and linkers useful for linking the VH and VL regions). Because anti-FLT 3 fragments (such as scFv fragments) that can be used in the extracellular domain of an anti-FLT 3 CAR are described elsewhere in the present application, this section specifically discusses only specific, non-limiting examples of anti-FLT 3 scFv. The scFv in the extracellular domain of the CAR enables the CAR to bind to target cells on their surface that express FLT3 (i.e., enables the CAR to bind to its target antigen).
In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO. 4 (or an amino acid sequence having at least 95% identity to SEQ ID NO. 4). In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO. 5 (or an amino acid sequence having at least 95% identity to SEQ ID NO. 5). In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO:44 (or an amino acid sequence having at least 95% identity to SEQ ID NO: 44). In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO. 45 (or an amino acid sequence having at least 95% identity to SEQ ID NO. 45). In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO. 46 (or an amino acid sequence having at least 95% identity to SEQ ID NO. 46). In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO:47 (or an amino acid sequence having at least 95% identity to SEQ ID NO: 47). In a specific embodiment, the anti-FLT 3 scFv comprises the amino acid sequence of SEQ ID NO:49 (or an amino acid sequence having at least 95% identity to SEQ ID NO: 49).
In some embodiments, the extracellular domain of the CAR comprises a signal peptide or a leader sequence. In some embodiments, the extracellular domain of the CAR comprises a cleavable signal peptide. In some embodiments, the extracellular domain of the CAR comprises a signal peptide prior to the anti-FLT 3 antigen binding domain (e.g., at the N-terminus of the antigen binding domain). Signal peptides are known in the art for use in CAR constructs. Some signal peptides help direct the nascent protein of the CAR to the endoplasmic reticulum. In some embodiments, the signal peptide is a GM-CSF signal peptide or an Igk chain signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO. 71. In some embodiments, the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 71. In some embodiments, the signal peptide comprises the nucleotide sequence of SEQ ID NO. 77. In some embodiments, the signal peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO 77.
In some embodiments, the linker connects the signal peptide to an anti-FLT 3 antigen binding domain (such as any anti-FLT 3 antigen binding fragment described herein, e.g., scFv). In some embodiments, the linker connects the signal peptide to an anti-FLT 3 light chain variable region (such as any anti-FT 3 VL described herein). In some embodiments, the linker connects the signal peptide to an anti-FLT 3 heavy chain variable region (such as any anti-FT 3 VH described herein). In some embodiments, the linker connecting the signal peptide to the antigen binding domain comprises 1-25 amino acids (e.g., 1, 2, 3, 4, or 5 amino acids), optionally comprising glycine and/or serine. In some embodiments, the linker connecting the signal peptide to the antigen binding domain is a diamino acid linker. In some embodiments, the linker that connects the signal peptide to the antigen binding domain is a glycine serine (e.g., GS) linker.
In some embodiments, the linker connects the extracellular domain to a spacer or hinge region. In some embodiments, the linker connecting the extracellular domain to the spacer or hinge region comprises 1-25 amino acids (e.g., 1,2, 3, 4, or 5 amino acids), optionally comprising glycine and/or serine. In some embodiments, the linker connecting the extracellular domain to the spacer or hinge region is a diamino acid linker. In some embodiments, the linker connecting the extracellular domain to the spacer or hinge region is a glycine serine (e.g., GS) linker.
Spacer or hinge region
In some embodiments, the extracellular domain is linked to the transmembrane domain by a hinge region or spacer. The hinge region may be any hinge region known in the art. Examples of hinge regions include, but are not limited to, hinge regions from CD8, CD28, or derived from IgG1, igG2, or IgG 4. In some embodiments, the hinge region is from the CD8 extracellular domain. In some embodiments, the hinge region is a CD8 (e.g., CD8 a) hinge. In some embodiments, the hinge region is a CD28 hinge. In some embodiments, the CD 8. Alpha. Hinge comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the CD 8a hinge comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 72. In some embodiments, the CD 8. Alpha. Hinge comprises the nucleotide sequence of SEQ ID NO: 78. In some embodiments, the CD 8a hinge comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 78.
Transmembrane domain
The transmembrane domain is a hydrophobic alpha helix that spans the cell membrane. For Chimeric Antigen Receptors (CARs), the transmembrane domain is capable of inserting the CAR into the cell membrane.
In some embodiments, the transmembrane domain is any one or more of the following: 4-1BB/CD137, activating NK cell receptor, immunoglobulin 、B7-H3、BALER、BLAME(SLAMF8)、BTLA、CD100(SEMA4D)、CD103、CD160(BY55)、CD18、CD19、CD19a、CD22、CD247、CD27、CD276(B7-H3)、CD28、CD29、CD3、CD3δ、CD3ε、CD3γ、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8、CD 8α、CD 8β、CD96(Tactile)、CDlla、CDllb、CDllc、CDlld、CD5、CD9、CD 16、CD33、CD37、CD64、CD80、CD86、CD134、CD137 or CD154 CEACAM1, CRT AM, CTLA4, PD-1, cytokine receptor, DAP-10, DNAMl (CD 226), fc gammase:Sub>A receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-, ig alphase:Sub>A (CD 79 ase:Sub>A), IL-2 Rbetase:Sub>A, IL-2 Rgammase:Sub>A, IL-7 Ralphase:Sub>A, inducible T cell costimulatory factor (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAMJTGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding CD83, LIGHT, LTBR, ly9 (CD 229) lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp, NKp44, NKp46, NKp80 (KLRFl), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD 162), signaling lymphocyte activating molecule (SLAM protein), SLAM (SLAMFl), SLAMF4 (CD 244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, toll ligand receptor, TRANCE/RANKL, VLA1 and VLA-6. In some embodiments, the transmembrane domain of a CAR provided herein is from a CD3 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a 4-1-BB transmembrane domain.
In some embodiments, the transmembrane domain of a CAR provided herein is a CD8 (e.g., CD8 a) transmembrane domain. In some embodiments, the transmembrane domain of a CAR provided herein is a CD3 transmembrane domain. In some embodiments, the transmembrane domain of a CAR provided herein is a CD4 transmembrane domain. In some embodiments, the transmembrane domain of a CAR provided herein is a CD28 transmembrane domain. In some embodiments, the transmembrane domain of a CAR provided herein is a 4-1-BB transmembrane domain. In some embodiments, the CD 8. Alpha. Transmembrane domain comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the CD 8. Alpha. Transmembrane domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO 73. In some embodiments, the CD 8. Alpha. Transmembrane domain comprises the nucleic acid sequence of SEQ ID NO. 79. In some embodiments, the CD 8. Alpha. Transmembrane domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 79.
Intracellular domain/intracellular domain
The intracellular domain of the CAR (i.e., the intracellular domain) is the functional end of the receptor. Following antigen recognition, the receptor aggregates and transmits a signal to the cell. The intracellular domain includes a signaling domain that transmits an internal signal to activate an immune cell expressing the CAR. In some embodiments, the intracellular domain of a CAR provided herein comprises a cd3ζ intracellular signaling (or activation) domain. The CD3 zeta signaling domain contains three immune receptor tyrosine based activation motifs (ITAMs). Upon binding of the antigen to the CAR, the ITAM transmits an activation signal to the CAR-containing cells.
In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO. 76. In some embodiments, the CD3 zeta signaling domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO 76. In some embodiments, the CD3 zeta signaling domain comprises the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, the CD3 zeta signaling domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 82.
In some embodiments, the intracellular domain of a CAR provided herein comprises a CD3 epsilon (epsilon) signaling domain. In some embodiments, the intracellular domain of a CAR provided herein comprises an fcγr (FcRgamma) signaling domain. Other activation domains known in the art may also be used.
In some embodiments, the intracellular domain comprises a co-stimulatory domain. In some embodiments, the intracellular domain comprises a stimulatory domain (e.g., a CD3 zeta signaling domain or another activating domain) and a co-stimulatory domain. In some embodiments, the co-stimulatory domain is a co-stimulatory domain of any one or more of: CD28, ICOS, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD 11 a/CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF 14), NKG2C, ig alpha (CD 79 a), DAP-10, fc gamma receptor, MHC class I molecules, TNF receptor proteins, immunoglobulins, cytokine receptors, integrins, SLAM proteins, ligands that activate NK cell receptors, BTLA, toll ligand receptor 、ICAM-1、B7-H3、CDS、ICAM-1、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL-2Rβ、IL-2Rγ、IL-7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD1 1a、LFA-1、ITGAM、CD1 1b、ITGAX、CD1 1c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRT AM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a、 specifically bind CD83, or any combination thereof.
In some embodiments, the intracellular domain comprises a CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO. 74. In some embodiments, the CD28 co-stimulatory domain comprises the nucleic acid sequence of SEQ ID NO. 80. In some embodiments, the CD28 costimulatory domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 80.
In some embodiments, the intracellular domain comprises a 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 75. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO 75. In some embodiments, the 4-1BB co-stimulatory domain comprises the nucleic acid sequence of SEQ ID NO. 81. In some embodiments, the 4-1BB costimulatory domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO. 81.
In some embodiments, the intracellular domain comprises both CD28 and 4-1BB costimulatory domains. In some embodiments, the intracellular domain comprises the amino acid sequences of SEQ ID NO 74 and SEQ ID NO 75.
In some embodiments, the intracellular domain comprises a co-stimulatory domain of CD 27. In some embodiments, the intracellular domain comprises a co-stimulatory domain of OX 40. In some embodiments, the intracellular domain comprises a costimulatory domain of ICOS. In some embodiments, the intracellular domain comprises a CD3 zeta signaling domain and a 4-1BB costimulatory domain. In some embodiments, the intracellular domain comprises a CD3 zeta signaling domain and a CD28 co-stimulatory domain. In some embodiments, the intracellular domain comprises a CD3 zeta signaling domain, a 4-1BB co-stimulatory domain, and a CD28 co-stimulatory domain.
In some embodiments, the intracellular domain comprises a CD3 zeta signaling domain having the amino acid sequence of SEQ ID NO. 76, a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO. 75, and a CD28 costimulatory domain having the amino acid sequence of SEQ ID NO. 74.
Safety switch
In some embodiments, the CAR comprises a safety switch. In some embodiments, the safety switch is selected from, but is not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 (icasp 9), and truncated human epidermal growth factor receptor (EGFRt) polypeptides. In some embodiments, the suicide gene is contained within a vector comprising a nucleic acid encoding any of the CARs described herein. In this way, administration of a prodrug designed to activate the safety switch (e.g., AP1903 that activates iCasp 9) triggers apoptosis of the safety switch and activates CAR-expressing cells.
In some embodiments, the suicide gene/safety switch is icasp9. In some embodiments, icasp9 achieves immune cell elimination (e.g., T cells) after administration of a dimerizing chemical inducer (e.g., AP1903 or AP 20187). In some embodiments, icasp9 is encoded by the nucleic acid sequence of SEQ ID NO. 85. In some embodiments, the icasp9 gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID No. 85. In some embodiments, the icasp9 safety switch comprises the amino acid sequence of SEQ ID NO. 105. In some embodiments, the icasp9 safety switch comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID No. 105.
In some embodiments, the suicide gene/safety switch is EGFRt. In some embodiments, EGFRt achieves immune cell depletion (e.g., T cells) after administration of an anti-EGFR monoclonal antibody (e.g., cetuximab). In some embodiments, EGFRt is encoded by the nucleic acid sequence of SEQ ID NO. 84. In some embodiments, the EGFRt gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO 84. In some embodiments, the EGFRt safety switch comprises the amino acid sequence of SEQ ID NO. 104. In some embodiments, the EGFRt safety switch comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO 104.
In some embodiments, a CAR containing a safety switch described herein comprises a self-cleaving peptide that connects the safety switch to the CAR. Exemplary self-cleaving peptides include 2A family peptides (e.g., T2A, E2A, F a and P2A peptides) and IRES. In some embodiments, the self-cleaving peptide is a T2A peptide. In some embodiments, the T2A peptide is encoded by the nucleic acid sequence of SEQ ID NO. 83. In some embodiments, T2A is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO 83. In some embodiments, the T2A peptide comprises the amino acid sequence of SEQ ID NO. 103. In some embodiments, the T2A peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO. 103.
In some embodiments, the self-cleaving peptide is an E2A peptide. In some embodiments, the self-cleaving peptide is an F2A peptide. In some embodiments, the self-cleaving peptide is a P2A peptide. In some embodiments, the self-cleaving peptide is an IRES peptide.
Isolated nucleic acids expressing CAR
Provided herein are nucleic acid sequences (polynucleotides) encoding one or more of the CARs provided herein. In some embodiments, the polynucleotide is contained within any vector suitable for transformation of immune cells (e.g., T cells). In some embodiments, the immune cells are transformed with a synthetic vector, a lentiviral vector, a retroviral vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, a lentivirus, an adenovirus, or a herpes virus).
Lentiviral vectors suitable for transforming T lymphocytes include, but are not limited to, for example, U.S. Pat. nos. 5,994,136;6,165,782;6,428,953;7,083,981; and 7,250,299, the disclosures of which are hereby incorporated by reference in their entirety. HIV vectors suitable for transforming T lymphocytes include, but are not limited to, vectors such as those described in U.S. Pat. No. 5,665,577, the disclosure of which is hereby incorporated by reference in its entirety.
In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 60. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 61. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 62. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 63. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 64. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 65. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 66. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 67. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 68. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 69. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 70.
In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 60. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 61. in some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 62. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 63. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 64. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 65. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 66. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 67. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 68. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 69. In some embodiments, the CAR comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% identity to SEQ ID No. 70.
In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 92. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 93. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 94. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 95. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 96. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 97. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 98. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 99. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 100. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 101. In some embodiments, the CAR is expressed from a plasmid of SEQ ID NO. 102.
Methods of making CARs
Methods of making CARs are well known in the art and are described, for example, in U.S. patent No. 6,410,319; U.S. patent No. 7,446,191; U.S. patent publication No. 2010/065818; U.S. Pat. nos. 8,822,647; PCT publication No. WO 2014/031687; U.S. patent No. 7,514,537; and Brentjens et al, 2007,Clin.Cancer Res.13:5426, each of which is hereby incorporated by reference in its entirety.
Binding of the extracellular antigen-binding domain of the CARs of the disclosure (e.g., the anti-FLT 3 antigen-binding fragments or scFv described herein) to FLT3 can be confirmed using methods known in the art. For example, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or western blot assay may be used. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by employing a labeling reagent (e.g., an antibody or scFv) that is specific for the complex of interest. For example, scfvs may be radiolabeled and used in Radioimmunoassays (RIA). The radioisotope may be detected by means such as using a y-counter or scintillation counter or by autoradiography. In some embodiments, the extracellular antigen binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include Green Fluorescent Protein (GFP), blue fluorescent protein (e.g., EBFP2, azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, cerulean and CyPet), and yellow fluorescent protein (e.g., YFP, citrine, venus and YPet). In some embodiments, the CAR is labeled with GFP. In some embodiments, the GFP-tagged CAR comprises the nucleic acid sequence of SEQ ID NO. 70. In some embodiments, the GFP-tagged CAR comprises the amino acid sequence of SEQ ID NO. 16.
Exemplary CAR constructs
In some embodiments, a CAR provided herein comprises the following domains:
Signal peptide-linker 1-VL-linker 2-VH-linker 3-hinge-TM domain-one or two co-stimulatory domains-signaling/activating domain. In some embodiments, the order of the domains is as specified herein. In some embodiments, any one or more of the linker domains are absent.
In some embodiments, a CAR provided herein comprises the following domains:
signal peptide-linker 1-VL-linker 2-VH-linker 3-hinge-TM domain-one or two co-stimulatory domains-signaling/activating domain-self-cleaving peptide-safety switch. In some embodiments, the order of the domains is as specified herein. In some embodiments, any one or more of the linker domains are absent.
In some embodiments, a CAR provided herein comprises the following domains:
the signal peptide-linker 1-VL-linker 2-VH-linker 3-CD8 hinge-CD 8TM-CD28 costimulatory domain and/or the 4-1BB costimulatory domain-CD 3 zeta signaling domain. In some embodiments, the order of the domains is as specified herein (but, for example, wherein the co-stimulatory domains (if all are present) occur in any order).
In some embodiments, a CAR provided herein comprises the following domains:
The signal peptide-linker 1-VL-linker 2-VH-linker 3-CD 8a hinge-CD 8a TM-CD28 costimulatory domain-4-1 BB costimulatory domain-CD 3 zeta signaling domain. In some embodiments, the order of the domains is as specified herein.
In some embodiments, in the CAR illustrated in this section:
VL is selected from the amino acid sequences comprising one of SEQ ID NOS.1, 2 and 28-38, and VH is selected from the amino acid sequences comprising one of SEQ ID NOS.3 and 17-27.
In some embodiments, a CAR provided herein comprises the following domains:
The signal peptide-linker 1-VL-linker 2-VH-linker 3-CD8 a hinge-CD 8 a TM-CD28 costimulatory domain-4-1 BB costimulatory domain-CD 3 zeta signaling domain; wherein the method comprises the steps of
(I) VL is selected from the amino acid sequences comprising one of SEQ ID NOS.1, 2 and 28-38,
(Ii) VH is selected from the amino acid sequences comprising one of SEQ ID NOs 3 and 17-27,
(Iii) Linker 2 comprises the sequence of SEQ ID NO. 53,
(Iv) The signal peptide comprises SEQ ID NO. 71,
(V) The CD8 alpha hinge comprises SEQ ID NO:72,
(Vi) CD 8. Alpha. TM. Contains SEQ ID NO:73,
(Vii) The CD28 co-stimulatory domain comprises SEQ ID NO 74,
(Viii) The 4-1BB co-stimulatory domain comprises SEQ ID NO 75, and
(Ix) The CD3 zeta signaling domain comprises SEQ ID NO 76.
In some embodiments, a CAR provided herein comprises: (i) An extracellular domain comprising any one of SEQ ID NOs 4, 5, 44, 45, 46, 47 and 49; (ii) a transmembrane domain; and (iii) an intracellular domain.
In some embodiments, a CAR provided herein comprises: (i) An extracellular domain comprising an scFv comprising the amino acid sequence of SEQ ID No. 4; (ii) a transmembrane domain; and (iii) an intracellular domain.
In some embodiments, a CAR provided herein comprises: (i) An extracellular domain comprising an scFv comprising the amino acid sequence of SEQ ID No. 5; (ii) a transmembrane domain; and (iii) an intracellular domain.
In some embodiments, the CAR comprises (i) an extracellular domain comprising an scFv comprising the amino acid sequence of SEQ ID NO:4, (ii) a transmembrane domain comprising a CD 8a transmembrane domain, and (iii) an intracellular domain comprising an intracellular signaling domain of cd3ζ and a costimulatory domain of CD28 and/or 4-1 BB.
In some embodiments, the CAR comprises (i) an extracellular domain comprising an scFv comprising the amino acid sequence of SEQ ID NO:5, (ii) a transmembrane domain comprising a CD 8a transmembrane domain, and (iii) an intracellular domain comprising an intracellular signaling domain of cd3ζ and a costimulatory domain of CD28 and/or 4-1 BB.
In some embodiments, the CAR comprises (i) an extracellular domain comprising an scFv comprising the amino acid sequence of SEQ ID NO:4, (ii) a transmembrane domain comprising a CD 8a transmembrane domain, (iii) an intracellular domain comprising an intracellular signaling domain of cd3ζ and a costimulatory domain of CD28 and/or 4-1BB, and (iv) a safety switch polypeptide.
In some embodiments, the CAR comprises (i) an extracellular domain comprising an scFv comprising the amino acid sequence of SEQ ID NO:5, (ii) a transmembrane domain comprising a CD 8a transmembrane domain, (iii) an intracellular domain comprising an intracellular signaling domain of cd3ζ and a costimulatory domain of CD28 and/or 4-1BB, and (iv) a safety switch polypeptide.
Exemplary CAR constructs are provided herein. In some embodiments, the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs 60-70. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 60. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 61. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 62. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 63. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 64. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 65. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 66. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 67. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 68. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 69. In some embodiments, the CAR comprises the nucleic acid sequence of SEQ ID NO. 70.
In some embodiments, the CAR comprises the amino acid sequence of any of SEQ ID NOs 6-16. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 6. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 7. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 8. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 9. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 10. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 11. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 12. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 13. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 14. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 15. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO. 16.
In some embodiments, the CAR (such as the CAR of SEQ ID NO: 16) is expressed from the plasmid depicted in FIG. 12A. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 7) is expressed from the plasmid depicted in FIG. 12B. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 8) is expressed from the plasmid depicted in FIG. 12C. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 6) is expressed from the plasmid depicted in FIG. 12D. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 12) is expressed from the plasmid depicted in FIG. 12E. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 11) is expressed from the plasmid depicted in FIG. 12F. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 10) is expressed from the plasmid depicted in FIG. 12G. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 9) is expressed from a plasmid depicted in FIG. 12H. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 13) is expressed from the plasmid depicted in FIG. 12I. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 14) is expressed from a plasmid depicted in FIG. 12J. In some embodiments, the CAR (such as the CAR of SEQ ID NO: 15) is expressed from the plasmid depicted in FIG. 12K.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 1, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 3.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID No.2, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 3.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 28, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 17.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:29, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:30, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:31, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:32, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:33, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 22.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:34, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 23.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 35, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 24.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:36, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 25.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 37, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 26.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) of a CAR described herein comprises (a) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:38, and (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO. 4. In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO. 44. In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO. 45. In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO. 46. In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO. 47. In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO: 49.
In some embodiments, the extracellular antigen-binding domain (e.g., scFV) of a CAR described herein comprises (a) a light chain variable region comprising an amino acid sequence ：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 and SEQ ID NO 38 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs; and (b) a heavy chain variable region comprising amino acid sequence ：SEQ ID NO:3、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID No. 27 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO.
In some embodiments, the extracellular antigen-binding domain (e.g., scFV) of a CAR described herein comprises (a) a light chain variable region comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:1, and (b) a heavy chain variable region comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3.
In some embodiments, an extracellular antigen-binding domain (e.g., scFV) comprises (a) a light chain variable region comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:2, and (b) a heavy chain variable region comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO. 4.
In some embodiments, the extracellular antigen-binding domain (e.g., scFv) comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO. 5.
CAR expressing immune cells
Provided herein are immune cells comprising (expressing) a CAR described herein. Provided herein are immune effector cells (e.g., T lymphocytes) comprising (expressing) a CAR described herein. Any immune cell having one or more effector functions, such as cytotoxic cell killing activity, cytokine secretion, induction of antibody directed cytotoxicity (ADCC), and/or Complement Dependent Cytotoxicity (CDC), may be used. In some embodiments, a CAR described herein is transduced, transfected or infected into an immune cell (e.g., a T cell).
In some embodiments, the immune cell is a T lymphocyte. T lymphocytes or "T cells" include, but are not limited to, thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments, the T cell is a helper T (Th) cell, such as a helper T1 (Th 1) or helper T2 (Th 2) cell. The T cells may be helper T cells (HTL; CD4 + T cells) CD4 + T cells, cytotoxic T cells (CTL; CD8 + T cells), CD4 +CD8+ T cells, CD4 -CD8- T cells, or any other T cell subset. Other exemplary T cell populations suitable for use in some embodiments include non-primed T cells and memory T cells. In some embodiments, the T lymphocytes are non-primed T lymphocytes or MHC restricted T lymphocytes. In some embodiments, the T lymphocytes provided herein are tumor-infiltrating lymphocytes (TILs).
In some embodiments, the immune cell is a natural killer cell (NK cell). In some embodiments, the immune cell is a NKT cell.
In some embodiments, the immune cell is a monocyte.
In some embodiments, the immune cell is a macrophage.
As the skilled artisan will appreciate, other cells may also be used as immune effector cells having a CAR as described herein.
In some embodiments, the immune cells are allogeneic. In some embodiments, the immune cells are autologous. In some embodiments, the immune cells are allogeneic T cells. In some embodiments, the immune cell is an autologous T cell. In some embodiments, the immune cells are obtained from a subject that is not the subject to be treated with the CAR-expressing immune cells.
In some embodiments, the immune cells are obtained from a healthy donor. In some embodiments, the immune cells are obtained from a patient suffering from cancer or tumor. In some embodiments, the immune cells are isolated from a tumor biopsy or expanded from immune cells isolated from a tumor biopsy. In some embodiments, the immune cells are isolated from, but are not limited to, bone marrow, fetal, neonatal, or adult or other hematopoietic cell sources, such as fetal liver, peripheral blood, lymph node tissue, thymus tissue, spleen tissue, or umbilical cord blood.
In some embodiments, the T lymphocytes are obtained from a healthy donor. In some embodiments, the T lymphocytes are obtained from a patient with cancer or tumor. In some embodiments, the T lymphocytes are obtained from a patient with cancer or tumor. In some embodiments, the T lymphocytes are isolated from a tumor biopsy or expanded from T lymphocytes isolated from a tumor biopsy. In some embodiments, the T cells are isolated from, but are not limited to, bone marrow, fetal, neonatal, or adult or other hematopoietic cell sources, such as fetal liver, peripheral blood, lymph node tissue, thymus tissue, spleen tissue, or umbilical cord blood.
Various techniques known in the art may be employed to isolate cells. Monoclonal antibodies are particularly useful for identifying markers associated with specific cell lineages and/or differentiation stages for positive and negative selection. Most terminally differentiated cells can initially be removed by relatively crude isolation. For example, magnetic bead separation may be used initially to remove large numbers of irrelevant cells. Separation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling with a cell density altering particle; performing magnetic separation by using magnetic beads coated with antibodies; affinity chromatography; cytotoxic agents used in conjunction or association with mabs, including but not limited to complement and cytotoxins; and panning with antibodies attached to a solid substrate, such as a plate, chip, panning, or any other convenient technique. Additional techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of complexity, e.g., multiple color channels, low angle and obtuse angle light scatter detection channels, impedance channels. Cells can be selected for dead cells by employing dyes associated with the dead cells, such as Propidium Iodide (PI).
In some embodiments, the disclosure provides an immune cell population comprising (e.g., expressing) a CAR as described herein (e.g., for treating cancer or conditioning prior to hematopoietic transplantation). For example, the population of immune cells can be obtained from Peripheral Blood Mononuclear Cells (PBMCs) of a patient (e.g., diagnosed with any of the cancers described herein) and modified to express a CAR described herein. PBMCs may be CD4 +、CD8+ or CD4 + and CD8 +.
The present disclosure provides methods for preparing immune cells expressing any of the CARs described herein. In some embodiments, the method comprises transfecting or transducing immune cells isolated from an individual such that the immune cells express one or more CARs as described herein. Methods for transfection, transduction and infection are well known in the art. In some embodiments, an immune cell described herein is transformed with a polynucleotide encoding a CAR described herein. In some embodiments, the T cell is transformed with a polynucleotide encoding a CAR described herein. In some embodiments, the immune cells described herein are expanded (i.e., proliferated) before and/or after transformation with a nucleic acid encoding a CAR described herein.
In some embodiments, immune cells are isolated from the individual and genetically modified to express the CAR without further in vitro manipulation, and then reapplied to the individual. In some embodiments, the immune cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express the CAR. Immune cells can be cultured or expanded prior to and/or after genetic modification (i.e., transduced or transfected to express a CAR contemplated herein).
In some embodiments, immune cells for autologous CAR therapy are prepared by: collecting leukocytes from a subject, isolating T cells from the leukocytes (e.g., using CD3/CD28 beads), transducing T cells with an anti-FLT 3 CAR (such as any CAR described herein), expanding the anti-FLT 3 CAR T cells, thereby producing a population of anti-FLT 3 CAR T cells useful for autologous CAR T therapy. Such cells may be infused into the same subject from which the primary white blood cells were obtained. In some embodiments, other immune cells are isolated from the subject in place of T cells, transduced with an anti-FLT 3 CAR, and expanded for autologous therapy.
In some embodiments, an immune cell (e.g., T cell) expresses about 1 to about 4, about 2 to about 4, about 3 to about 4, about 1 to about 2, about 1 to about 3, or about 2 to about 3 vector copies per cell of a CAR described herein.
In some embodiments, an immune cell (e.g., a T cell) expresses a CAR comprising the nucleic acid sequence of any one of SEQ ID NOs 60-70. In some embodiments, immune cells (e.g., T cells) express a CAR comprising the amino acid sequence of any of SEQ ID NOs 6-16. In some embodiments, an immune cell (e.g., a T cell) expresses a CAR comprising a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of SEQ ID NOS: 60-70. In some embodiments, an immune cell (e.g., a T cell) expresses a CAR comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of SEQ ID NOS.6-16.
In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 60. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 61. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 62. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 63. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 64. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 65. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 66. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 67. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 68. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 69. In some embodiments, the T cell expresses a CAR comprising the nucleic acid sequence of SEQ ID NO. 70. In any of these embodiments, another immune cell may be used in place of a T cell, such as an NK cell, macrophage or monocyte.
In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 7. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 8. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 9. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 10. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 12. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 13. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 14. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 15. In some embodiments, the T cell expresses a CAR comprising the amino acid sequence of SEQ ID NO. 16. In any of these embodiments, another immune cell may be used in place of a T cell, such as an NK cell, macrophage or monocyte.
In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising an scFv of the amino acid sequence of any of SEQ ID NOs 4, 5, 44, 45, 46, 48, or 49. In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising the scFv of SEQ ID NO. 4. In some embodiments, an immune cell (e.g., a T cell) expresses any CAR described herein, comprising the scFv of SEQ ID No. 5. In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising the scFv of SEQ ID NO: 44. In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising the scFv of SEQ ID NO: 45. In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising the scFv of SEQ ID NO: 46. In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising the scFv of SEQ ID NO: 47. In some embodiments, an immune cell (e.g., a T cell) expresses any of the CARs described herein, comprising the scFv of SEQ ID NO: 49.
Pharmaceutical composition
Provided herein are pharmaceutical compositions comprising (i) any anti-FLT 3 antibody or fragment described herein (e.g., any scFv described herein) or any immune cell (including a population of immune cells) that expresses an anti-FLT 3CAR described herein, and (ii) a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers, including but not limited to excipients and stabilizers, are known in the art (see, e.g., remington's Pharmaceutical Sciences (1990) Mack Publishing co., easton, PA).
In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, isotonic agents, buffers, suspending agents, dispersing agents, emulsifying agents, wetting agents, complexing agents, chelating agents, pH buffering agents, solubilizing agents, antioxidants, anesthetics, and/or antibacterial agents. In some embodiments, the carrier is selected from, but not limited to, one or more of the following: water, brine, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, starch, lactose, sucrose, gelatin, malt, propylene, silica gel, sodium stearate, and dextrose, and combinations thereof. In some embodiments, the pharmaceutically acceptable carrier further comprises auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding protein.
In some embodiments, when administered parenterally, the pharmaceutically acceptable carrier includes, but is not limited to, physiological saline or Phosphate Buffered Saline (PBS), solutions containing agents such as glucose, polyethylene glycol, polypropylene glycol, or other agents.
In some embodiments, the pharmaceutical composition is formulated to provide rapid, sustained or delayed release of the active ingredient after administration. Formulations for providing rapid, sustained or delayed release of an active ingredient after administration are known in the art (Mishra,M.K.(2016).Handbook of encapsulation and controlled release.Boca Raton,CRC Press,Taylor&Francis Group,CRC Press under the brand name Informa flag company Taylor & Francis Group, incorporated herein by reference in its entirety).
In some embodiments, the pharmaceutical compositions provided herein comprise any anti-FLT 3 antibody or fragment described herein (e.g., any scFv described herein) or any anti-FLT 3 CAR expressing immune cell described herein (including immune cell populations) and one or more other therapeutic agents (e.g., anti-cancer agents) in a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition is formulated for administration to a subject by any route. In some embodiments, the pharmaceutical composition is formulated for injection and prepared as a liquid solution, suspension, emulsion, or solid form suitable for making into a solution or suspension prior to injection.
In some embodiments, an anti-FLT 3 antibody or fragment described herein (e.g., any scFv described herein) or an immune cell (including an immune cell population) expressing an anti-FLT 3 CAR described herein in a pharmaceutical composition is present in a therapeutically effective amount. The therapeutically effective amount is determined by methods known in the art.
Therapeutic method
Cancer treatment
In some embodiments, the disclosure provides methods for treating cancer comprising administering any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein.
In some embodiments, the method of treating cancer comprises administering to a subject any anti-FLT 3 antibody or fragment described herein or any immune cell described herein that expresses an anti-FLT 3 CAR that binds to a FLT3 epitope of a cell (e.g., a FLT3 epitope of a target cell). In some embodiments, a method of treating cancer comprises administering to a subject any anti-FLT 3 antibody or fragment described herein or any anti-FLT 3 CAR expressing immune cell described herein that binds to a FLT3 epitope of a cancer cell (e.g., an AML cell).
In some embodiments, the present disclosure provides a method of treating a cancer that is resistant to one or more other cancer therapies (e.g., vaccine, chemotherapy, radiation therapy, small molecule therapy, or immunotherapy (such as treatment with another antibody).
The methods described herein are suitable for treating cancers that are expected, known or determined to express FLT3 on their cell surfaces.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein is administered according to a method described herein to achieve or result in one or more of the following: (i) a decrease in the frequency or number of cancer cells, (ii) a decrease in the growth of cancer or an increase in the number of cancer cells, (iii) an inhibition of the progression of cancer cell growth, (iv) a regression of cancer, (v) an inhibition of recurrence of cancer, (vi) an eradication of cancer, (vii) a decrease or improvement in the severity or duration of one or more symptoms of cancer, (viii) an inhibition of the development or onset of one or more symptoms associated with cancer, (ix) an enhancement or improvement in the therapeutic effect of another anti-cancer therapy, (x) an increase in the life expectancy or survival of the subject, (xi) a decrease in hospitalization (e.g., hospitalization) of the subject, (xii) an improvement in the quality of life of the subject, (xiii) a decrease in mortality, and (xiv) an increase in the survival or remission of the subject without recurrence. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein is administered according to a method described herein to achieve or result in a reduction in tumor burden in a subject (e.g., an effective reduction in tumor burden relative to the tumor burden in a subject prior to treatment).
In some embodiments, administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is effective to treat cancer (e.g., reduce the frequency or number of cancer cells, reduce the growth or proliferation of cancer cells, increase the life expectancy or survival, eradicate the cancer, or ameliorate one or more symptoms of cancer) in a subject when used alone or in combination with another therapy.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject is effective to reduce the frequency or number of, or eliminate, cancer cells. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject is effective to reduce the number or frequency of cancer cells by at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cancer cell level of the subject prior to administration of the therapy). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of cancer cells by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cancer cell level of the subject prior to administration of the therapy). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of cancer cells by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cancer cell level of the subject prior to administration of the therapy). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of cancer cells by at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the level of cancer cells in the subject prior to administration of the therapy).
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3CAR described herein to a subject is effective to treat any cancer (e.g., AML) described herein. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3CAR described herein to a subject is effective to slow the progression of any cancer (e.g., AML) described herein. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3CAR expressing immune cell described herein to a subject is effective to reduce tumor burden of any cancer (e.g., AML) described herein. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3CAR expressing immune cell described herein to a subject is effective to increase the survival of a subject having any cancer described herein. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3CAR expressing immune cell described herein is effective to increase the median survival of the subject relative to a subject untreated or treated with a placebo. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3CAR expressing immune cell described herein is effective to increase the median survival of the subject relative to a subject treated with standard of care therapy.
Examples of cancer cells that may be reduced in number or eliminated using the methods described herein include, but are not limited to, blast cells of Acute Myeloid Leukemia (AML), blast or leukemic blast cells of Acute Lymphoblastic Leukemia (ALL), blast cells of Chronic Myeloid Leukemia (CML), blast plasmacytoid dendritic cell tumor (BPDCN), and blast cells of Chronic Lymphocytic Leukemia (CLL).
In certain embodiments, an immune cell expressing any anti-FLT 3 CAR described herein is used in a method of treating a subject described herein. In some embodiments, the immune cells expressing the anti-FLT 3 CAR are autologous to the subject being treated. In some embodiments, blood (e.g., white blood cells) is collected (e.g., by apheresis) from a subject, then immune cells (e.g., T cells) are isolated from the blood (e.g., using anti-CD 3/CD28 beads), then nucleic acid encoding an anti-FLT 3 CAR is introduced into the isolated immune cells (optionally then isolated immune cells comprising the anti-FLT 3 CAR can be expanded), and then the immune cells comprising the anti-FLT 3 CAR thus obtained are administered (e.g., by infusion) to the subject (i.e., the same subject from which the immune cells were isolated). Such autologous CAR T therapy is depicted in fig. 2A. In other embodiments, the immune cells expressing the anti-FLT 3 CAR are not autologous to the subject being treated.
Hematopoietic cell conditioning
In some embodiments, the present disclosure provides methods for preparing or conditioning a subject in need thereof for hematopoietic cell transplantation. In some embodiments, the subject in need thereof is a patient who is eligible, about to receive, or is receiving a Bone Marrow (BM) hematopoietic stem cell and/or hematopoietic progenitor cell transplantation. In some embodiments, a subject in need of hematopoietic cell transplantation suffers from cancer (such as any of the cancers described herein).
In some embodiments, the present disclosure provides methods for preparing or conditioning a subject in need thereof for hematopoietic cell transplantation, wherein any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein is administered to the subject.
In some embodiments, the method of preparing or conditioning a subject comprises administering to the subject any anti-FLT 3 antibody or fragment described herein or any anti-FLT 3CAR expressing immune cell described herein that binds to a FLT3 epitope on hematopoietic stem cells. In some embodiments, the method of preparing or conditioning a subject comprises administering to the subject any anti-FLT 3 antibody or fragment described herein or any anti-FLT 3CAR expressing immune cell described herein that binds to a FLT3 epitope on a hematopoietic cell. In some embodiments, the method of preparing or conditioning a subject comprises administering to the subject any anti-FLT 3 antibody or fragment described herein or any anti-FLT 3CAR expressing immune cell described herein that binds to a FLT3 epitope on a dendritic cell. In some embodiments, the method of preparing or conditioning a subject comprises administering to the subject any anti-FLT 3 antibody or fragment described herein or any anti-FLT 3CAR expressing immune cell described herein that binds to a FLT3 epitope on a myeloid cell. In some embodiments, the method of preparing or conditioning a subject comprises administering to the subject any anti-FLT 3 antibody or fragment described herein or any anti-FLT 3CAR expressing immune cell described herein that binds to a FLT3 epitope on lymphoid cells.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to condition the subject prior to hematopoietic cell transplantation.
In certain embodiments, an immune cell expressing any anti-FLT 3 CAR described herein is used in a method of treating a subject described herein. In some embodiments, the immune cells expressing the anti-FLT 3 CAR are autologous to the subject being treated. In some embodiments, blood is collected from a subject, then immune cells (e.g., T cells) are isolated from the blood, then nucleic acid encoding an anti-FLT 3 CAR is introduced into the isolated immune cells (optionally then isolated immune cells comprising an anti-FLT 3 CAR can be expanded), and then the thus obtained autoimmune cells comprising an anti-FLT 3 CAR are administered to the subject. In other embodiments, the immune cells expressing the anti-FLT 3 CAR are not autologous to the subject being treated.
In some embodiments, an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein is effective to significantly reduce or eliminate the frequency or number of cells of Hematopoietic Stem Cells (HSCs) and/or Hematopoietic Progenitor Cells (HPCs) (e.g., early hematopoietic progenitor cells). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of HSCs and/or HPCs (e.g., early HPCs) by at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of HSCs and/or HPCs (e.g., early HPCs) by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of HSCs and/or HPCs (e.g., early HPCs) by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of HSCs and/or HPCs (e.g., early HPCs) by at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy). In some of these embodiments, the reduction in HSCs and/or HPCs (e.g., early HPCs) occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells).
In some embodiments, an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein is effective to significantly reduce or eliminate the frequency or number of cells of a pluripotent progenitor cell (MPP) and/or a common progenitor Cell (CP). In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of MPPs and/or CPs relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of MPPs and/or CPs relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of MPPs and/or CPs relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject is effective to reduce the number or frequency of MPPs and/or CPs by at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy). In some of these embodiments, the decrease in MPP or CP occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells).
According to some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject is effective to condition a patient undergoing Bone Marrow (BM) HSC and/or HPC (e.g., early HPC) transplantation. In some embodiments, the subject receives a HSC transplant. In some embodiments, the subject receives HPC transplantation. In some embodiments, the subject receives HSC and HPC (e.g., early HPC) transplants. In some embodiments, the subject receives MPP and/or CP. In some embodiments, HSC/HPC transplantation is used to treat any hematological cancer described herein, e.g., acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), dendritic cell tumors, and the like.
In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of myeloid lineage (e.g., circulating myeloid lineage cells or monocytes). In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of myeloid lineage cells (e.g., circulating myeloid lineage cells or monocytes) by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy). In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of myeloid lineage cells (e.g., circulating myeloid lineage cells or monocytes) by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy).
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject does not significantly reduce the number or frequency of myeloid lineage cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of bone marrow lineage cells by less than 60%, less than 55%, less than 50%, less than 40%, less than 30%, or less than 20% relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy).
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject reduces the number or frequency of myeloid lineage cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of bone marrow lineage cells by at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy).
In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces a population of cells that express one or more (e.g., one, two, three, four, five, or six) of CD45, FLT3, CD19, CD38, CD33, and CD 34. In some of these embodiments, the reduction in the population of cells expressing one or more (e.g., one, two, three, four, five, or six) of CD45, FLT3, CD19, CD38, CD33, and CD34 occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in the population of cells expressing one or more (e.g., one, two, three, four, five, or six) of CD45, FLT3, CD19, CD38, CD33, and CD34 occurs in the circulating blood cells (e.g., in bone marrow mononuclear cells) of the subject being treated.
In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces a population of cells expressing one or more (e.g., one, two, three, or four) of FLT3, CD38, CD33, and CD 34. In some of these embodiments, the reduction in the population of cells expressing one or more (e.g., one, two, three, or four) of CD45, FLT3, CD19, CD38, CD33, and CD34 occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in the population of cells expressing one or more (e.g., one, two, three, or four) of CD45, FLT3, CD19, CD38, CD33, and CD34 occurs in circulating blood cells (e.g., in bone marrow mononuclear cells) of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 60%. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 70%. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 80%. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells by at least 90% relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy). In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of FLT3 expressing cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 95%. In some of these embodiments, the reduction in FLT3 expressing cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in FLT3 expressing cells occurs in circulating blood cells of the subject being treated. In some of these embodiments, the decrease in FLT3 expressing cells is a decrease in cancer cells in the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+ hematopoietic stem cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+ hematopoietic stem cells relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction in cd34+ hematopoietic stem cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in cd34+ hematopoietic stem cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of early hematopoietic progenitor cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of early hematopoietic progenitor cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction of early hematopoietic progenitor cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction of early hematopoietic progenitor cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an immune cell expressing an anti-FLT 3 CAR described herein to a subject reduces the number or frequency of dendritic cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of dendritic cells by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% relative to a control or baseline (e.g., relative to the cellular level of the subject prior to administration of the therapy). In some of these embodiments, the reduction in dendritic cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in dendritic cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd45+cd19+ cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd45+cd19+ cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd45+cd19+ cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by about 55%, about 50%, about 45%, about 40%, or about 35% (or between about 30% and 55%). In some of these embodiments, the reduction in cd45+cd19+ cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in cd45+cd19+ cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+cd38+ cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+cd38+ cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction in cd34+cd38+ cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in cd34+cd38+ cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+cd38-cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+cd38-cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd34+cd38-cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction in cd34+cd38-cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in cd34+cd38-cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the population of cells expressing CD34 by at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99%.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the population of cells expressing FLT3 by at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99%.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd33+ cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd33+ cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the population of cells expressing CD33 by at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99%. In some of these embodiments, the reduction in CD33 expressing cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in CD33 expressing cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd1c+ myeloid dendritic cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd1c+ myeloid dendritic cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction in cd1c+ myeloid dendritic cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in cd1c+ myeloid dendritic cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd141+ myeloid dendritic cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of cd141+ myeloid dendritic cells relative to a control or baseline (e.g., relative to the level of cells of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction in cd141+ myeloid dendritic cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in cd141+ myeloid dendritic cells occurs in circulating blood cells of the subject being treated.
In some embodiments, administering an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein to a subject reduces the number or frequency of CD303 + plasmacytoid dendritic cells. In some embodiments, administration of an anti-FLT 3 antibody or fragment described herein, a pharmaceutical composition described herein, or an anti-FLT 3 CAR expressing immune cell described herein reduces the number or frequency of CD303 + plasmacytoid dendritic cells relative to a control or baseline (e.g., relative to the cell level of the subject prior to administration of the therapy) by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%. In some of these embodiments, the reduction in CD303 + plasmacytoid dendritic cells occurs in the bone marrow of the subject being treated (e.g., in bone marrow mononuclear cells). In some of these embodiments, the reduction in CD303 + plasmacytoid dendritic cells occurs in the circulating blood cells of the subject being treated.
In some embodiments, the method of HSC/HPC transplantation comprises transplantation of donor HSC/HPC cells. In some embodiments, the donor cell is from a healthy subject. In other embodiments, the HSC/HPC transplantation includes transplantation of autologous cells (e.g., obtained prior to the onset of the disease being treated).
In some embodiments, the present disclosure provides methods of performing hematopoietic stem/progenitor cell transplantation in a subject, comprising:
(i) Reducing the number of Hematopoietic Stem Cells (HSCs) and/or Hematopoietic Progenitor Cells (HPCs) by administering to a subject an anti-FLT 3 antibody or fragment as described herein, a pharmaceutical composition as described herein, or an immune cell expressing an anti-FLT 3 CAR as described herein,
(Ii) HSCs/HPCs (e.g., donor HSCs/HPCs) are transplanted into a subject.
In some embodiments, the present disclosure provides methods of performing hematopoietic stem/progenitor cell transplantation in a subject, comprising:
(i) Reducing the number of Hematopoietic Stem Cells (HSCs) and/or Hematopoietic Progenitor Cells (HPCs) by administering to a subject a population of immune cells expressing a CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs 6 and 9-15,
(Ii) HSCs/HPCs (e.g., donor HSCs/HPCs) are transplanted into a subject.
Cancer to be treated
Cancers may be treated according to the methods described herein. In some embodiments, the cancer to be treated is a hematopoietic cancer or a hematologic cancer. Examples of hematological cancers treated according to the methods described herein include, but are not limited to, acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Myeloid Leukemia (CML), chronic Lymphoblastic Leukemia (CLL), blast plasmacytoid dendritic cell tumor (BPDCN), peripheral T cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, neuroblastoma, non-malignant hereditary or acquired myelopathy, multiple myeloma, and dendritic cell tumor. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is Acute Myeloid Leukemia (AML). In some embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL). In some embodiments, the cancer is Chronic Myeloid Leukemia (CML). In some embodiments, the cancer is Chronic Lymphocytic Leukemia (CLL). In some embodiments, the cancer is a blast plasmacytoid dendritic cell tumor (BPDCN). In some embodiments, the cancer is peripheral T cell lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is hodgkin's lymphoma. In some embodiments, the cancer is non-hodgkin's lymphoma. In some embodiments, the cancer is a neuroblastoma. In some embodiments, the cancer is a non-malignant genetic or acquired bone marrow disorder. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a dendritic cell tumor.
In some embodiments, the cancer is the result of a non-malignant genetic or acquired bone marrow disorder. Examples of non-malignant genetic or acquired bone marrow disorders treated according to the methods described herein include, but are not limited to, sickle cell anemia, severe beta thalassemia, refractory Diamond-black fan anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia, paroxysmal sleep hemoglobinuria, pure red cell aplastic anemia, fanconi anemia, megakaryocytopenia, and congenital thrombocytopenia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is sickle cell anemia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is severe beta thalassemia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is refractory Diamond-Blackfan anemia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is myelodysplastic syndrome. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is idiopathic severe aplastic anemia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is paroxysmal nocturnal hemoglobinuria. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is pure red blood cell aplastic anemia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is fanconi anemia. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is megakaryocytoma. In some embodiments, the non-malignant genetic or acquired bone marrow disorder is congenital thrombocytopenia.
Application method
Any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein, can be administered to a subject by any suitable means, including, but not limited to, parenteral (e.g., intravenous, intra-arterial, intramuscular, intra-osseous, intra-brain, intrathecal, subcutaneous), intraperitoneal, intratumoral, intrapulmonary, intradermal, transdermal, conjunctival, intraocular, intranasal, intratracheal, oral, and topical intralesional routes of administration. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein (where reference to an immune cell also includes a population of such immune cells) is administered intravenously, intra-arterially, intraperitoneally, or intratumorally.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered intravenously (e.g., by bolus injection or continuous infusion). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered intraperitoneally. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered intramuscularly. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered subcutaneously. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered intratumorally (such as by injection into a tumor of the cancer being treated). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered intravenously, intraperitoneally, or intratumorally.
Various dosing regimens of anti-FLT 3 antibodies and fragments described herein, pharmaceutical compositions described herein, and immune cells expressing an anti-FLT 3 CAR described herein are contemplated, including single administration or multiple administrations over a period of time. Methods of administration include, but are not limited to, bolus administration, pulsed infusion, and continuous infusion.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein is administered one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more times. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered once. In some embodiments, when administered once intravenously (e.g., without additional repeated administrations), any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein is effective in the methods described herein.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered once every about 1 to 7 days for about 1 to 8 weeks. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered once every about 1 to 7 days for about 1 to 4 weeks. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered once every about 3 to 7 days for about 2 to 3 weeks. In some embodiments, the administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is once every about 3 days for about 2 weeks to once every about 7 days for about 3 weeks. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered once every about 2 to 4 days for about 2 to 3 weeks (e.g., 2 weeks or 3 weeks).
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered weekly for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days (e.g., weekly, twice weekly, every other day, or daily). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered for less than 6 weeks, less than 5 weeks, less than 4 weeks, less than 3 weeks, or less than 2 weeks. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered at a frequency of once every two days or less (e.g., for 1 to 3 weeks). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered at a frequency of once every three days or less (e.g., for 1 to 3 weeks). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered once every four days or less frequently (e.g., for 1 to 3 weeks). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered at a frequency of once every five days or less (e.g., for 1 to 3 weeks). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3CAR expressing immune cell described herein is administered at a frequency of once per week or less (e.g., for 1 to 3 weeks).
In some embodiments, administration (of an antibody, fragment, composition, or immune cell described herein) is once every 3 days for about 2 weeks. In some embodiments, administration is once every 4 days for about 2 weeks. In some embodiments, administration is once every 5 days for about 2 weeks. In some embodiments, administration is once every 7 days for about 2 weeks. In some embodiments, administration is once every 3 days for about 3 weeks. In some embodiments, administration is once every 4 days for about 3 weeks. In some embodiments, administration is once every 5 days for about 3 weeks. In some embodiments, administration is once every 7 days for about 3 weeks.
In some embodiments, administration is once weekly for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, administration is twice weekly for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, administration is three times per week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, administration is four times per week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, administration is five times per week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, administration is six times per week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, administration occurs seven times per week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.
In some embodiments, the administration is weekly, biweekly, tricyclically, biweekly, pentacyclic, or hexaweekly. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, twelve, thirteen, fifteen, seventeen, eighteen, nineteen, or twenty (e.g., during treatment).
Administration as described herein includes regimens in which an initial dose of any of the therapies described herein is followed by one or more lower doses, or in which the initial dose is followed by one or more higher doses. In some embodiments, the initial dose is followed by one or more lower doses. In some embodiments, the initial dose is followed by one or more higher doses.
In some embodiments, an initial treatment period (wherein any of the therapies described herein are administered, e.g., once a month, once a week, twice a week, or three times a week) is followed by a withdrawal period during which no therapy is administered (for example, one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months, or one year), followed by a second treatment period (wherein the therapy is administered, e.g., once a month, once a week, twice a week, or three times a week). Such an initial treatment period and such a second treatment period may last for example two weeks, three weeks, four weeks, six weeks (where the initial treatment period may be the same or different from the second treatment period). The course of treatment (with initial treatment period, withdrawal period, and second treatment period) may be repeated twice, three times, four times, five times, six times, ten times, or more than ten times.
In some embodiments, a therapeutically effective amount of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject or patient. The therapeutically effective amount will depend on the method used, the cancer being treated, the severity of the cancer being treated, the route of administration, the target site, the condition of the patient (e.g., age, weight, health), the responsiveness of the patient, other medications used by the patient, and other factors considered by the physician performing the treatment, as appropriate.
In some embodiments, an immune cell (e.g., T cell) expressing an anti-FLT 3 CAR is expressed in about 1x10 6, about 5x10 6, about 1x10 7, about 2x10 7, About 3x10 7, about 4x10 7, about 5x10 7, about 6x10 7, about 7x10 7, About 8x10 7, about 9x10 7, about 1x10 8, about 2x10 8, about 3x10 8, about 4x10 8, about 5x10 8, about 6x10 8, about 7x10 8, about 8x10 8, About 9x10 8, about 1x10 9, about 2x10 9, about 3x10 9, about 4x10 9, About 5x10 9, about 6x10 9, about 7x10 9, about 8x10 9, about 9x10 9, About 1x10 10, about 2x10 10, about 3x10 10, about 4x10 10, or about 5x10 10 cells.
In some embodiments, an immune cell (e.g., T cell) expressing an anti-FLT 3 CAR is expressed in about 5x10 7, about 6x10 7, about 7x10 7, about 8x10 7, About 9x10 7, about 1x10 8, about 2x10 8, about 3x10 8, about 4x10 8, About 5x10 8, about 6x10 8, about 7x10 8, about 8x10 8, about 9x10 8, About 1x10 9, about 2x10 9, about 3x10 9, about 4x10 9, about 5x10 9, About 6x10 9, about 7x10 9, about 8x10 9, about 9x10 9, or 1x10 10 cells.
In some embodiments, immune cells (e.g., T cells) expressing an anti-FLT 3 CAR are administered in an amount of about 1x10 8, about 2x10 8, about 3x10 8, about 4x10 8, about 5x10 8, about 6x10 8, about 7x10 8, about 8x10 8, about 9x10 8, about 1x10 9, about 2x10 9 cells.
In some embodiments, immune cells (e.g., T cells) expressing an anti-FLT 3 CAR are administered in an amount of about 5x10 7 to about 1x10 10 cells. In some embodiments, immune cells (e.g., T cells) expressing an anti-FLT 3 CAR are administered in an amount of about 1x10 8 to about 2x10 9 cells.
In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is from about 0.01mg/kg to about 10mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.01mg/kg to about 2mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.05mg/kg to about 1mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.1mg/kg to about 0.5mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.1mg/kg to about 0.3mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.01mg/kg, about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, or about 2mg/kg of patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.1mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.2mg/kg patient body weight. In some embodiments, the dose of any anti-FLT 3 antibody or fragment described herein is about 0.3mg/kg patient body weight.
In some embodiments, hematopoietic cell transplantation occurs 5 days to 5 weeks after administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein. In some embodiments, the hematopoietic cell transplantation occurs about 2 to 3 weeks after administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein. In some embodiments, the hematopoietic cell transplantation is performed about 1 to 4 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed about 10 to 25 days after administration. In some embodiments, the hematopoietic cell transplantation is performed about 10 to 20 days after administration. In some embodiments, the hematopoietic cell transplantation is performed about 2 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed about 3 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed at least 5 days or 1 week after administration. In some embodiments, the hematopoietic cell transplantation is performed at least 2 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed less than 3 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed less than 4 weeks after administration. In some embodiments, the hematopoietic cell transplantation is performed less than 5 weeks after administration.
Patient population
In some embodiments, the patient or subject is treated with any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein. In some embodiments, the patient or subject is a mammal, such as a human, non-human primate, dog, cat, rabbit, cow, horse, goat, sheep, or pig. In some embodiments, the subject is a human.
In some embodiments, a patient or subject treated according to the methods described herein has (e.g., has been diagnosed as having) cancer. Methods for cancer diagnosis are known in the art. In some embodiments, the cancer is an early stage cancer. In some embodiments, the cancer is advanced cancer.
In some embodiments, a patient or subject treated according to the methods described herein has (e.g., has been diagnosed with) a hematopoietic cancer or hematological cancer. In some embodiments, the hematological cancer is Acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), chronic Myelogenous Leukemia (CML), blast plasmacytoid dendritic cell tumor (BPDCN), peripheral T cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, multiple myeloma, non-malignant genetic or acquired myelogenous disorder, or dendritic cell tumor.
In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with Acute Myeloid Leukemia (AML). In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with Acute Lymphoblastic Leukemia (ALL). In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with Chronic Lymphocytic Leukemia (CLL). In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with Chronic Myeloid Leukemia (CML). In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with a blast plasmacytoid dendritic cell tumor (BPDCN). In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with a peripheral T cell lymphoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with follicular lymphoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with diffuse large B-cell lymphoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with hodgkin's lymphoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with non-hodgkin's lymphoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with a neuroblastoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with multiple myeloma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with a dendritic cell tumor.
In some embodiments, a patient or subject treated according to the methods described herein has (e.g., has been diagnosed with) a non-malignant inherited acquired bone marrow disorder. In some embodiments, the non-malignant hereditary acquired myelopathy is sickle cell anemia, severe beta thalassemia, refractory Diamond-Blackfan anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia, paroxysmal sleep hemoglobinuria, pure red blood cell aplastic anemia, fanconi anemia, megakaryocytopenia, congenital thrombocytopenia, or Severe Combined Immunodeficiency (SCID).
In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with sickle cell anemia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with severe beta thalassemia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with refractory Diamond-Blackfan anemia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with myelodysplastic syndrome. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with idiopathic severe aplastic anemia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed as having paroxysmal sleep hemoglobinuria. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with pure red blood cell aplastic anemia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed as having fanconi anemia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with megakaryocytoma. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with congenital thrombocytopenia. In some embodiments, a patient or subject treated according to the methods described herein has been diagnosed with Severe Combined Immunodeficiency (SCID).
In some embodiments, the patient or subject being treated has previously received one or more cancer therapies (e.g., vaccine, small molecule targeted therapy, chemotherapy, radiation therapy, or immunotherapy) and has developed resistance to one or more previous cancer therapies. In some embodiments, the patient or subject being treated is resistant to chemotherapy. In some embodiments, the patient or subject being treated is resistant to the small molecule targeted therapy. In some embodiments, the patient or subject being treated is resistant to another immunotherapy.
In some embodiments, the patient or subject has a type of cancer known or expected to express FLT3 on its cell surface.
In some embodiments, the patient or subject being treated has a cancer that has been determined to express FLT3 on its cell surface using methods known in the art, which FLT3 can be targeted by any anti-FLT 3 antibody or fragment described herein or any immune cell expressing an anti-FLT 3 CAR described herein.
In some embodiments, a patient or subject treated according to the methods described herein is in need of hematopoietic cell transplantation. In some embodiments, a patient or subject treated according to the methods described herein is in need of bone marrow transplantation with hematopoietic stem cells and/or hematopoietic progenitor cells.
Combination therapy and kit
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject in combination with one or more anti-cancer therapies. In some embodiments, the anti-cancer therapy is chemotherapy, surgery, radiation therapy, antibody therapy, small molecule therapy, or another anti-cancer therapy known in the art.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject in combination with radiation therapy. Examples of the types of chemotherapeutic agents that may be used in the methods described herein include, but are not limited to, alkylating agents, nitrosourea agents, antimetabolites, topoisomerase inhibitors, aromatase inhibitors, antitumor antibiotics, plant-derived alkaloids, hormone antagonists, P-glycoprotein inhibitors, and platinum complex derivatives. Specific examples of chemotherapeutic agents useful in the methods described herein include, but are not limited to, paclitaxel (taxol), paclitaxel (paclitaxel), albumin-bound taxol, 5-fluorouracil (5-FU), gemcitabine (gemcitabine), daunorubicin (daunorubicin), colchicine (colchicin), mitoxantrone (mitoxantrone), tamoxifen (tamoxifen), cyclophosphamide, nitrogen mustard, busulfan (busulfan), uramending (uramustine), Mustard root (mustargen), ifosfamide (ifosamide), bendamustine (bendamustine), carmustine (carmustine), lomustine (lomustine), semustine (semustine), fotemustine (fotemustine), streptozocin (streptozocin), thiotepa (thiotepa), mitomycin (mitomycin), filigree quinone (diaziquone), tetrazine, altretamine, mitozolomide (mitozolomide), and pharmaceutical compositions, Temozolomide, procarbazine (procarbazine), hexamethylmelamine, altretamine, kemaline (hexalen), trefosfamide (trofosfamide), estramustine (estramustine), busulfan (treosulfan), mannsulfane (mannosulfan), triamine quinone (triaziquone), carboquinone (carboquone), nimustine (nimustine), ramustine (ranimustine), azathioprine (azathioprine), sulfanilamide, fluoropyrimidine, thiopurine, thioguanine, mercaptopurine, cladribine (cladribine), capecitabine (capecitabine), pemetrexed (pemetrexed), fludarabine (fludarabine), hydroxyurea, nelarabine (nelarabine) or clofarabine (clofarabine), cytarabine, decitabine (decitabine), pravastatin (pralatrexate), Fluorouridine, thioguanine, azacytidine (azacitidine), cladribine (cladribine), penstatin, mercaptopurine, imatinib (imatinib), actinomycin D (dactinomycin), selobidine (cerubidine), actinomycin (actinomycin), flavomycin (luteomycin), epirubicin (epirubicin), idarubicin (idarubicin), plicamycin (plicamycin), pramoxine, vincristine (vincristin), vinorelbine (vinorelbine), vinflunine (vinflunine), paclitaxel, docetaxel (docetaxel), etoposide (etoposide), teniposide (teniposide), vinca (periwinkle), vinca (vinca), taxane, irinotecan (irinotecan), topotecan (topotecan), camptothecin, teniposide, pirarubicin (pirarubicin), neomycin (novobiocin), Mylabris (merbarone), azithromycin (aclarubicin), amsacrine (amsacrine), antiandrogens, antiestrogens, bicalutamide (bicalutamide), medroxyprogesterone, fluoromethylprogesterone, diethylstilbestrol, estrace, octreotide (octreotide), megestrol (megestrol), raloxifene (raloxifene), toremifene (toremifene), fulvestrant (fulvestrant), prednisone (prednisone), Fluotamide (flutamide), leuprorelin (leuproolide), goserelin (goserelin), aminoglutethimide (aminoglutethimide), testosterone (testolactone), anastrozole (anastrozole), letrozole (letrozole), exemestane (exemestane), vorozole (vorozole), formestan (formestane), method Qu (fadrozole), androstene, resveratrol (resveratrol), tricresyl (Texazole), Creatinine, catechin (catachin), apigenin (apigenin), eriodictyol (eriodictyol), isoliquiritigenin (isoliquiritigenin), mangostin (mangostin), amiodarone (amiodarone), azithromycin (azithromycin), captopril (captopril), clarithromycin (clarithromycin), cyclosporine (cyclosporine), piperine (piperine), quercetin (quercetine), Quinidine (quinidine), quinine (quinine), reserpine (reserpine), ritonavir (ritonavir), tarquasimide (tariquidar), verapamil (verapamil), cisplatin, carboplatin, oxaliplatin (oxaliplatin), antiplatin (transplatin), nedaplatin (nedaplatin), satraplatin (satraplatin), triplatin (triplatin), and carboplatin.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject in combination with one or more anti-tumor agents selected from the group consisting of: anthracyclines (e.g., daunorubicin (daunomycin) and doxorubicin (doxorubicin)), auristatin (auristatin), methotrexate (MTX), vindesine (vindesine), neocarcinomycin (neocarzinostatin), cisplatin, chlorambucil (chlorambucil), cytarabine (cytosine arabinoside), 5-fluorouridine, melphalan (melphalan), ricin (ricin) and calicheamicin (calicheamicin), including combination chemotherapeutics such as with doxorubicin, bleomycin (bleomycin), vinca (vinblastine) and dacarbazine (ABVD), BEACOPP or upgraded BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) and stam fuv (doxorubicin, vinca, nitrogen mustard, vincristine, laimycin, etoposide and prednisone). In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject in combination with one or more of the following: immunotherapy (e.g., anti-CD 20 antibody rituximab), immunotoxins (e.g., vitamin b-rituximab (Brentuximab vedotin) (SGN-35), which is an immunotoxin consisting of CD-30 directed antibodies linked to the anti-tubulin agent monomethyl auristatin E (MMAE)), adoptive immunotherapy (cytotoxic T lymphocytes), programmed death 1 (PD-1) blockade (e.g., nano Wu Liyou mab (nivolumab), pamplybuzumab (pembrolizumab)).
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject having a cancer (e.g., AML or ALL) in combination with a chemotherapeutic agent specified for the cancer, which chemotherapeutic agent may optionally be administered at an administration dose and/or regimen specified for the cancer.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject in combination with an immunotherapy. In some embodiments, the immunotherapy comprises administration of a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD 1 antagonist, an anti-PD-L1 antagonist, and an anti-CTLA 4 antagonist. In some embodiments, the checkpoint inhibitor is an anti-PD 1 antagonist. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody (such as an antagonistic anti-PD-1 antibody). In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antagonist. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody (such as an antagonistic anti-PD-L1 antibody). In some embodiments, the checkpoint inhibitor is an anti-CTLA 4 antagonist (e.g., an antagonistic anti-CTLA 4 antibody). In some embodiments, the checkpoint inhibitor is a Lag3 antagonist. In some embodiments, the checkpoint inhibitor is a Tim3 antagonist. In some embodiments, the checkpoint inhibitor is a TIGIT antagonist. In some embodiments, the checkpoint inhibitor is an OX40 antagonist.
In some embodiments, the anti-PD 1 antagonist is selected from the group consisting of, but not limited to: nalmefene Wu Liyou mab, palbociclizumab, PDR001, pembrolizumab (Pembrolimumab) (Bio X Cell), bio X Cell clone J116 (catalog No. BE 0188), cimipran Li Shan mab (cemiplimab), and pidilizumab. In some embodiments, the anti-PD-L1 antagonist is selected from the group consisting of, but not limited to: atilizumab (atezolizumab), avimab (avelumab), dulcis You Shan anti (durvalumab), yw243.55.s70, MPDL3280A, MDX-1105 and BMS-936559. In some embodiments, the anti-CTLA 4 antagonist is selected from, but not limited to, ipilimumab (ipilimumab) and tremelimumab (tremelimumab).
In some embodiments, an immune cell comprising an anti-FLT 3 CAR described herein (such as an anti-FLT 3 CAR having the amino acid sequence of any of SEQ ID NOs: 6 and 9-15 below), or an immune cell comprising any of the nucleic acid sequences SEQ ID NOs: 60 and 63-69 below, is administered to a subject in combination with chemotherapy. In some embodiments, any anti-FLT 3 antibody or fragment having a VH and/or VL described herein or any scFv described herein is administered to a subject in combination with chemotherapy.
In some embodiments, an immune cell comprising an anti-FLT 3 CAR described herein (such as an anti-FLT 3 CAR having the amino acid sequence of any of SEQ ID NOs: 6 and 9-15 below), or an immune cell comprising any of the nucleic acid sequences SEQ ID NOs: 60 and 63-69 below, is administered to a subject in combination with an immunotherapy (e.g., a checkpoint inhibitor). In some embodiments, any anti-FLT 3 antibody or fragment having a VH and/or VL described herein or any scFv described herein is administered to a subject in combination with an immunotherapy (e.g., a checkpoint inhibitor).
In some embodiments, an immune cell comprising an anti-FLT 3CAR described herein (such as an anti-FLT 3CAR having the amino acid sequence of any of SEQ ID NOs: 6 and 9-15 below), or an immune cell comprising any of the nucleic acid sequences SEQ ID NOs: 60 and 63-69 below, is administered to a subject in combination with an anti-PD 1 antagonist (e.g., an anti-PD 1 antibody). In some embodiments, an immune cell comprising an anti-FLT 3CAR described herein (such as an anti-FLT 3CAR having the amino acid sequence of any of SEQ ID NOs: 6 and 9-15 below), or an immune cell comprising any of the nucleic acid sequences SEQ ID NOs: 60 and 63-69 below, is administered to a subject in combination with an anti-PD-L1 antagonist (e.g., an anti-PDL 1 antibody). In some embodiments, an immune cell comprising an anti-FLT 3CAR described herein (such as an anti-FLT 3CAR having the amino acid sequence of any of SEQ ID NOs: 6 and 9-15 below), or an immune cell comprising any of the nucleic acid sequences SEQ ID NOs: 60 and 63-69 below, is administered to a subject in combination with an anti-CTLA 4 antagonist (e.g., an anti-CTLA 4 antibody). In some embodiments, any anti-FLT 3 antibody or fragment having a VH and/or VL described herein or any scFv described herein is administered to a subject in combination with any anti-PD-1 antagonist, any anti-PD-L1 antagonist, or any anti-CTLA 4 antagonist described herein.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject in combination with radiation therapy (e.g., X-ray, gamma-ray, electron beam).
In some embodiments, the checkpoint inhibitor is administered prior to administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein. In some embodiments, the checkpoint inhibitor is administered concurrently with the administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein. In some embodiments, the checkpoint inhibitor is administered after administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein.
In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to the subject between, during, or after the second therapy.
In some embodiments, a subject treated according to the methods described herein does not receive an anti-cancer therapy prior to administration of any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3 CAR described herein. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein, is administered to a subject that has received an anti-cancer treatment prior to administration of the antibody or fragment. In some embodiments, any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any anti-FLT 3 CAR expressing immune cell described herein is administered to a subject recovering from or receiving immunosuppressive therapy.
In some embodiments, provided herein are kits comprising any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3CAR described herein, and one or more additional anti-cancer agents. In some embodiments, provided herein are kits comprising (i) any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3CAR described herein (e.g., in a therapeutically effective amount), and (ii) one or more chemotherapeutic agents (e.g., in a therapeutically effective amount, which may be less than the therapeutic amount of the one or more agents when the antibody, fragment, or immune cell is not used). In some embodiments, provided herein are kits comprising (i) any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3CAR described herein (e.g., in a therapeutically effective amount), and (ii) one or more checkpoint inhibitors described herein (e.g., in a therapeutically effective amount, which may be less than the therapeutic amount of the one or more drugs when the antibody, fragment, or immune cell is not used). In some embodiments, provided herein are kits comprising (i) any anti-FLT 3 antibody or fragment described herein, any pharmaceutical composition described herein, or any immune cell expressing an anti-FLT 3CAR described herein (e.g., in a therapeutically effective amount), and (ii) one or more anti-PD 1 antibody, anti-PD-l 1 antibody, or anti-CTLA 4 antibody (e.g., in a therapeutically effective amount, which may be less than the therapeutic amount of the one or more drugs when the antibody, fragment, or immune cell is not used).
The following examples are provided by way of illustration and not limitation. Various other embodiments of the invention may be practiced in view of the general description provided herein.
Examples
Example 1: humanized antibodies and single chain variable fragment generation.
Prior to humanization, chimeric anti-FLT 3 antibodies were evaluated for competitive binding to FLT3 ligand (FLT 3L). Specifically, REH cells were incubated with 10nM recombinant human FLT3L (R & Dsystems) for 20 min and washed with PBS+2% BCS+2mM EDTA (running buffer). The cells were then stained with different concentrations of chimeric monoclonal antibodies prepared in running buffer. Cells were washed five times with running buffer and stained with anti-human IgG Fc antibody conjugated to Alexa Fluor 488 (Jackson Immunoresearch Laboratories, 109-545-008). Cells were stained with 7-AAD (7-AAD viability staining solution, biolegend 420404) and then analyzed by flow cytometry. The binding of chimeric antibodies 1-18BA (comprising mouse VL (SEQ ID NO: 28) and mouse VH (SEQ ID NO: 17) and human IgG) was not reduced by FLT3L pretreatment, but was substantially the same as in the case where FLT3L pretreatment was not used, as shown in FIG. 1A. This suggests that 118BA does not compete with FLT3L for binding to FLT3.
To generate the humanized antibodies and single-chain variable fragments described herein, the following methods were used.
Materials and methods
Variable domain analysis and CDR identification
For the purpose of identifying CDRs and analyzing the closest matching germline sequences, IMGT domain gap alignment tools were used: http_www_imgt. Org/3 Dstructure-DB/cgi/domainGapAlig.
Molecular modeling
Molecular models were constructed for VH and VL domains using software based on previously published homology of antibody crystal structures.
Gene synthesis and cloning
The variable heavy and variable light domains (for FLT 3) are designed with appropriate restriction sites at the 5 'and 3' ends to enable cloning into Absolute Antibody cloning and expression vectors. The variable domain sequences are codon optimized for expression in human cells. After gene synthesis, the variable domains are cloned into Absolute Antibod y vectors of the appropriate species and type. The correctness of the sequence was verified by sanger sequencing (Sanger sequencing) and analyzing the raw data using DNASTAR LASER GENE software. Once confirmed, plasmid DNA preparation of the appropriate size is performed to generate sufficient quantities of high quality DNA for transfection.
Expression and purification
Following plasmid generation, HEK 293 (human embryonic kidney 293) mammalian cells were passaged to the optimal stage of transient transfection. Cells were transiently transfected with heavy and light chain expression vectors and cultured for an additional 6 days. Cultures were harvested by centrifugation at 4000rpm and filtered through a 0.22 μm filter. The first step of purification was performed by protein a affinity chromatography eluting with citrate pH 3.0 buffer followed by neutralization with 0.5m tris, pH 9.0. The eluted protein buffer was then exchanged into PBS using a desalting column. The antibody concentration was determined by UV spectroscopy and the antibody was concentrated as required.
Antibody analysis
Antibody purity was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and HPLC (high performance liquid chromatography). SEC-HPLC was performed on an Agilent 1100 series instrument using an appropriate Size Exclusion Column (SEC). Antibody expression titers were determined by protein a HPLC.
Humanization
Sequence analysis
The VH and VL sequences of 1-18BA (SEQ ID NO:17 and SEQ ID NO:28, respectively) were generated using the methods described in U.S. patent publication No. 20190389955 (the entire contents of which are incorporated herein by reference) and run through IGMT gap alignment tool to analyze for all known antibody germline sequences. CDR regions are specified using IMGT definition. The alignment of sequences with muse is most clear, especially IGHV8-8 family of VH and IGKV9-124 of VL.
Molecular modeling
To achieve structure-directed humanization, models were created for the 1-18BA murine VH and VL sequences.
Germ line selection
The VH and VL sequences were aligned to the Absolute Antibody database of human germline sequences. Table 1 shows germline sequences selected as humanized frameworks.
Table 1. Heavy and light chain germline sequences selected as humanized frameworks and their percent identity to the original murine VH and VL sequences.
CDR grafting
For humanization of the antibodies, the VH and VL sequences were run by a CDR grafting algorithm to transfer CDRs from murine antibody 1-18BA (VH with SEQ ID NO:17 and VL with SEQ ID NO: 28) onto selected human germline sequences. Although CDRs are defined as being primarily responsible for binding to antigen, amino acids outside of these regions (referred to as framework regions) may be directly involved in binding or play a role in correctly positioning the CDRs. A structure-directed approach was used to determine which framework amino acids remained the same as the original mouse amino acids to preserve binding integrity. Table 2 summarizes the sequences generated.
TABLE 2 original mouse and humanized sequences generated for 1-18BA
Sequence responsibility analysis
To ensure that highly undesirable sequence responsibilities are not introduced into the humanized sequence, the original mouse sequence and humanized sequence were run through Absolute Antibody sequence responsibilities tool. The most interesting sequences are responsible for the glycosylation motif and the free cysteines. The original parent VH sequence contains an N-linked glycosylation motif. The motif is located within CDR-H2 and may have an effect on binding. Most humanized VH sequences retain this motif except for cAb 1981.
Antibody production and analysis
Antibody cloning
As described above, a total of 4 humanized heavy chains and 3 humanized light chains were designed. Each of these was synthesized separately and cloned into human IgG1 and scFv. At the time of transfection, all possible combinations of humanized sequences were performed to generate a total of 12 different humanized IgG and 12 humanized scFv.
Antibody expression and purification
All antibodies were expressed on a small scale and the proteins were purified by protein a or nickel chromatography. All purified products appeared to be expected under non-reducing and reducing SDS-PAGE. All IgG's were expressed well except cAB 1984-10.0. scFv showed mixed expression levels, of which 6 failed to be fully expressed (cAb 1977, cAb1081, cAb1982, cAb1983, and cAb 1988). IgG and scFV yields are shown in tables 3 and 4.
Table 3 shows the tables of chimeric human IgG and humanized IgG. The VH and VL protein sequences are shown as percent identity to human germline sequences, titer, amount of final purified antibody and monomer content.
Table 4 shows a table of mouse scFv and humanized scFv. The protein sequence is shown as well as the percent identity to the human germline sequence, titer, and amount of final purified scFv.
Aggregation analysis
Purified IgG was analyzed for aggregation and fragmentation by SEC-HPLC. The monomer content is reported in table 3. All antibodies showed over 95% monomer purity, and most antibodies showed over 98% monomer purity.
Example 2: novel discovery of fully humanized anti-FLT 3 IgG clone and its binding affinity to scFv
This example shows that humanized anti-FLT 3 antibodies, such as the antibodies described herein, have high binding affinity for FLT 3. In particular, this example shows that humanized anti-FLT 3IgG and anti-FLT 3 scFv have high binding affinity to FLT3 expressing cells.
Fully humanized anti-human FLT3 monoclonal IgG1, clone human 1-18BA-v1, clone ID cAb1978-30.11, antibodies (VL with SEQ ID NO:1 and VH with SEQ ID NO: 3) and His-tagged scFv, clone human 1-18BA-v1, antibodies with the same VH and VL as IgG1 (comprising SEQ ID NO:4 and His tag at the C-terminus) were developed as described above. The binding affinity of the scFv molecules was compared to IgG1 monoclonal molecules using REH cells highly expressing FLT3 (acute lymphoblastic leukemia cell line, ATCC accession number CRL-8286). REH cells were incubated in triplicate for 30 min with different concentrations (10 -1 to 10 4 ng/mL) of IgG1 or scFv molecules diluted in FACS buffer (hereinafter simply "buffer", phosphate Buffered Saline (PBS) (Caisson Labs, accession number PBL 06) +2% BCS (GE HEALTHCARE, accession number SH 30073.04) +1mM EDTA (Invitrogen, accession number 15575020)) at 4℃in a final volume of 100. Mu.L. Cells were washed 3 times with buffer and incubated with secondary antibody. IgG1 was detected with a 1:200 goat anti-human Fc FITC (Jackson) secondary antibody, and scFv was detected with an anti-His FITC (1:200) secondary antibody. The secondary antibodies were incubated in triplicate in 100 μl final volume for 30 min at 4 ℃. Cells were washed once with buffer and resuspended in 200uL buffer+1:100 7-AAD viability staining solution (Biolegend, accession number 420404) and analyzed by flow cytometry. Flow cytometry acquisitions were performed using Beckman Coulter CytoFLEX (Beckman Coulter) and analysis was performed using FlowJo (TREESTAR INC, ashland, OR). The graph shows the median fluorescence intensity of REH cells as a function of the concentration of primary antibody used. Variable slope (four parameters) curves were fitted to the data and the EC50 was used to compare binding affinities. The EC50 of the IgG1 molecule was 62.1ng/mL (0.41 nM) and the EC50 of the scFv molecule was 85.45ng/mL (3.42 nM) (FIGS. 1B-1C).
Example 3: generation of third generation anti-FLT 3 chimeric antigen receptor
To generate the CAR, a lentiviral vector encoding a third generation CAR under transcriptional control of the EF-1 a promoter was used (figure 2B). The CAR encodes a polypeptide comprising the anti-FLT 3 scFv sequence described in example 1 (SEQ ID NO: 4), followed by CD28 and 4-1BB costimulatory domain, followed by CD3 zeta activation domain. The CAR sequence is followed by the CopGFP sequence linked by a self-cleaving T2A sequence (SEQ ID NO: 16). The vector allows for dual expression of CARs and CopGFP from a single RNA transcript. All constructs were verified by sequencing.
FLT3 is expressed on Hematopoietic Stem and Progenitor Cells (HSPCs) and dendritic cells, and in acute myeloid leukemia. When T cells are exposed to FLT3 + target cells, expression of the CAR in the T cells allows for MHC independent primary activation via the anti-FLT 3 scFv. This activation results in the expression of perforin and granzyme by the co-stimulatory and activation domains in the CAR, which ultimately produces cytotoxicity to the target cells (fig. 2C).
Example 4: isolation and transduction of T cells
To generate anti-FLT 3 CAR T cells, the following protocol was performed. This example shows that anti-FLT 3 CAR T cells have been successfully generated, that expression of anti-FLT 3 CAR T cells peaks 4 days after transduction, and that T cells expressing anti-FLT 3 CAR expand approximately 125-fold within 18 days.
On day 1, T cells were isolated from adult peripheral blood (purchased from New York Blood Center, NYBC) using a negative magnetic separation kit (StemCELL Technologies, no. 17951). T cells were mixed with Dynabeads (human T activator CD3/CD28, gibco, accession number 111.61D) at a 1:1 cell to bead ratio and inoculated at a density of 8X 10 4 cells/well OR 1.6X10 5 cells/well into 200ul medium (RPMI 1640 medium (ATCC, accession number 30-2001) +10% heat inactivated Fetal Bovine Serum (FBS) (Biowest, accession number S1620) +1% penicillin/streptomycin (Gibco, accession number 15140122)) in untreated 96-well flat bottom cell culture plates OR 10 6 cells/well into 1mL medium in 24 well plates on day 2, T cells were transduced with T lentiviral vectors using 1:1000 polybrene (5 mg/mL) (VectorBuilder) at several MOI (0, 2, 5, 10 and 20) on day 4, the transduction efficiency was determined by flow cytometry, GFP positive cells were successfully transduced, surface expression of scFv was confirmed using polyclonal anti-Fab APC antibody (Jackson ImmunoResearch, accession No. 109-607-003) T cells were stained with scFv detection antibody at 4℃for 30 min, cells were washed once with buffer and resuspended in 200. Mu.L buffer and analyzed by flow cytometry, flow cytometry was performed using Beckman Coulter CytoFLEX (Beckman Coulter) and analysis was performed using FlowJo (TREESTAR INC, ashland, OR), T cell medium was changed and recombinant human IL-2 (Miltenyi Biotec, accession No. 130097745) was supplemented at a final concentration of 10ng/mL dividing T cells as required to maintain cell densities between 0.5 and 1X 10 6 cells/mL on day 7, transduction efficiency was assessed by flow cytometry as described above. On day 10, transduction efficiency was assessed by flow cytometry as described above, and T cells were ready for functional cytotoxicity testing (fig. 3A).
CAR T cells were generated and expanded as described above. Specifically, CAR transduced cells with the amino acid sequence of SEQ ID NO. 16 (domains with the following orientation: signal peptide-linker-scFV-linker-CD 8. Alpha. Hinge-CD 8. Alpha. Transmembrane domain-CD 28-41BB-CD3 zeta-T2A-GFP) were used. Transduction efficiencies were determined at different MOI over a period of 10 days. Specifically, GFP expression in cells transduced with anti-FLT 3 CAR3a (SEQ ID NO: 16) was measured. For all MOIs, GFP expression peaked on day 4, did not drop until day 7, and appeared to be stable on day 10 (fig. 3B). When transduced with anti-FLT 3 CAR3a, T cells expanded approximately 125-fold within 18 days (fig. 3C). As expected, expression of GFP (i.e., expression of CAR constructs) appears to be linearly related to expression of scFv (fig. 3D).
Example 5: in vivo and in vitro anti-FLT 3CAR-T cytotoxicity against AML cell line MOLM-13
This example shows that the anti-FLT 3 CAR described in example 4 is effective against AML cell lines in vitro and effectively increases the survival of leukemia animal models in vivo.
CAR T cells were generated and expanded as described above and then used in functional cytotoxicity assays. Specifically, cytotoxicity of cells was measured using an AML cell line expressing FLT3, such as MOLM-13 (DSMZ, accession No. ACC 554) labeled using CELLTRACE TM Violet cell proliferation kit (Thermo FISHER SCIENTIFIC, accession No. C34571) (fig. 4A). CAR T cells (effector cells) expressing CAR encoded by SEQ ID NO. 16 (encoding the domains in the following directions: signal peptide-linker-SEQ ID NO. 4) scFV-linker-CD 8. Alpha. Hinge-CD 8. Alpha. Transmembrane domain-CD 28-41BB-CD3 ζ -T2A-GFP) were co-cultured with labeled AML cells (target cells) at different effector cell to target cell ratios (10:1, 5:1, 2:1, 1:1, 1:2 and 1:5) for 24 and 48 hours. Cells were harvested, washed with FACS buffer, and resuspended in 200. Mu.L of 11:100 7-AAD viability staining solution (Biolegend, accession number 420404) and analyzed by flow cytometry. Flow cytometry acquisitions were performed using Beckman Coulter CytoF LEX (Beckman Coulter) and analysis was performed using FlowJo (TREES TAR INC, ashland, OR). Representative dot plots show flow cytometry data after debris removal. Target cells were identified as CellTraceViolet + and effector cells were identified as CellTraceViolet -. CellTraceViolet + cells were gated to determine the frequency of dead (7 AAD +) and viable (7 AAD -) target cells. Cytotoxicity to MOLM-13 was significantly higher when co-cultured with anti-FLT 3 CAR3a-T cells for 24 hours and 48 hours in vitro compared to control T cells (fig. 4B and 4C).
The in vivo efficacy of anti-FLT 3CAR 3a-T cells against leukemia was evaluated in female nod.cg-Prkdc scid Il2rgtm1Sug/JicTac (abbreviated as NOG mice, taconic, numbered NOG-F hereinafter) transplanted with an AML cell MOLM-13 cell line (DSMZ, numbered ACC 554) transduced to express EGFP. For each NOG mouse (n=14), 2×10 5 EGFP-MOLM-13 cells were transplanted intravenously on day 1. On days 5 and 32, each mouse received 4×10 6 control T cells (n=7) or 4×10 6 anti-FLT 3 CAR3a T cells (33% CAR +) (n=7). Some mice received 4×10 6 CAR-T cells (33% CAR +) (n=4) to evaluate the effect of CAR-T alone on mice (i.e., anti-FLT 3 CAR3a T cells without MOLM-13 cells) (fig. 5A). mice exhibiting physiological distress, cachexia or hind leg paralysis were sacrificed. Compared to 24 days in control T cell mice, anti-FLT 3 CAR3a-T cell treatment extended median survival to 47 days (fig. 5B). Mice were bled every two weeks after T cell administration. Peripheral blood mononuclear cells were treated with anti-mouse CD45 APC (BioLegend, accession number 103112), anti-human CD45 APC-eFluor780 (Invitrogen, accession number 47045942), anti-human CD33 BV510 (BioLegend, accession number 366610), and anti-human CD3 PE-Cy7 (BioLegend, Number 300420) and analyzed by flow cytometry to determine the frequency of MOLM-13 cells (mCD 45 -hCD 45+CD33+EGFP+) and T cells (mCD 45 -hCD45+CD3+) or CAR-T cells (mCD 45 -hCD45+CD3+EGFP+) in the circulation. flow cytometry acquisitions were performed using Beckman Coulter CytoFLEX (Beckman Coulter) and analysis was performed using FlowJo (TREESTAR INC, ashland, OR). In treated animals, the frequency of T cells in anti-FLT 3 CAR3a-T cells (relative to the frequency of total monocytes) was maintained to day 47, and in some cases to day 72, whereas control T cell treated mice died at day 28 (fig. 5C-5E).
Example 6: targeting CD34 + bone marrow HSPC with FLT3-CAR-T cells for Hematopoietic Stem Cell Transplantation (HSCT) conditioning
This example shows that anti-FLT 3 CAR T cells described herein effectively achieve depletion of cd34+ HSPCs. This demonstrates the possibility that anti-FLT 3 CAR T cells are used for HSCT.
Preparation and transplantation of human HSC
Mononuclear cells (MNCs) from fresh Cord Blood (CB) units (Carolina Cord Blood Bank) were isolated by density centrifugation using Ficoll separation medium (StemCELL Technologies, no. 07861). To further purify MNC, erythrocytes were lysed using lysis buffer (ALFA AESAR, no. J62150-AP). Human CD34 + cells in CB MNC were then enriched using anti-human CD34 microbeads (Miltenyi Biotec, accession No. 130-046-703). T cells in the CD34 - fraction of MNC were enriched using negative magnetic separation (StemCELL Technologies, no. 17951).
3-4 Week old NOG female mice were injected with 2.4X10 5 CD34 + cells and 10 5 T cells. Blood was drawn from the submaxillary vein of the mice every 4 weeks (about 100 μl) to assess human chimerism (fig. 6A). PBMCs were stained using the following mAb groups to determine the level of humanization and lineage development: anti-mouse CD45 APC (BioLegend, accession No. 103112), anti-human CD45 APC-eFluor780 (Invitrogen, accession No. 47045942), anti-human CD3 PE-Cy7 (BioLegend, accession No. 300420), anti-human CD19 PE (BioLegend, accession No. 302208), anti-human CD33 FITC (BioLegend, accession No. 303304), anti-human CD4 BV605 (BioLegend, 317438), anti-human CD8 BV510 (BioLegend, accession No. 344732), anti-human CD45RA BV650 (BioLegend, accession No. 304136), anti-human CD45RO Pacific blue (BioLegend, accession No. 304216).
Conditioning Using autologous CAR-T cells
When the above mice (fig. 6A) showed robust human implantation (> 1% human CD45 +), at 27 weeks post-transplantation, the mice received 5×10 6 autologous control T cells (expanded in the same manner as CAR-T cells) or 5×10 6 autologous CAR-T cells (expressing the CARs described in example 3). Mice were bled 4 days, 14 days, and 18 days after T cell therapy. Mice were euthanized on day 18 after treatment with T cells and peripheral blood and Bone Marrow (BM) were isolated. The overall frequency of human CD45 + cells in the peripheral blood MNC fraction before and after treatment with control or CAR-T cells was measured and shows similar engraftment between control T cells and anti-FLT 3CAR T cells (fig. 6B). Lineage frequencies (T cells (CD 3 +), B cells (CD 19 +) and myeloid cells (CD 33 +)) were measured in peripheral blood before and after treatment with control or CAR-T cells (fig. 6C), and changes in frequency over time were measured (fig. 6D). Mice treated with anti-FLT 3 CAR-T cells showed a significant decrease in myeloid cells compared to control T cell treated mice. The change in cell frequency in isolated Bone Marrow (BM) was then measured. The overall anatomical differences in the femur and tibia of the mice were assessed visually and showed no difference between control T cells and animals treated with anti-FLT 3 CAR T cells (fig. 7A). MNC (BM-MNC) total cell counts from BM were recorded. BM-MNC was also analyzed by flow cytometry as described above and the frequency of human CD45 + cells was determined (FIG. 7B). Lineage frequencies (T cells (CD 3 +), B cells (CD 19 +) and myeloid cells (CD 33 +)) were measured in BM-MNCs (fig. 7C). The lineage cell counts (T cells (CD 3 +), B cells (CD 19 +) and myeloid cells (CD 33 +)) of individual mice in both cohorts before and after treatment with control or CAR-T cells are shown (fig. 7D). In CAR-T treated mice, cd45+cd19+ B cell counts were in a decreasing trend (54.4% decrease compared to control). Without being bound by theory, B cell counts may decrease due to a decrease in the number of HSCs, MPPs, and CPs.
BM-MNCs were stained using the following mAb panel to determine HSPC frequency: anti-mouse CD45 APC (BioLegend, accession number 103112), anti-human CD45 APC-eFluo r780 (Invitrogen, accession number 47045942), anti-human lineage mixture BV510 (BioLege nd, accession number 348807), anti-human CD34 PE-Cy7 (BioLegend, accession number 343516), anti-human CD38 FITC (BioLegend, accession number 356610), anti-human CD90 PE (Invitr ogen, accession number 12090942), and anti-human CD45RA BV650 (BioLegend, accession number 304136). Significant depletion of hematopoietic stem and progenitor cells (CD 38 +CD34+ and CD38 -CD34+ populations) was observed in FLT3-CAR T treated mice compared to controls (fig. 8A). Progenitor cells in bone marrow of CAR T cell treated mice were significantly reduced compared to control mice. Further gating on the CD38 -CD34+ population revealed significant depletion of "true" Hematopoietic Stem Cells (HSCs) (CD 90 +CD45RA-), multipotent progenitor cells (MPPs) (CD 90 -CD45RA-) and common progenitor Cells (CPs) (CD 90 -CD45RA+) (fig. 8A and 8B).
Example 7: generation of CAR constructs with suicide switches
A CAR construct incorporating a suicide safety switch was generated. In particular, EGFRt (truncated EGFR), an Epidermal Growth Factor Receptor (EGFR) -based safety switch, is co-expressed on a CAR plasmid following the T2A peptide sequence (the resulting CAR has the amino acid sequence of SEQ ID NO:7 and comprises the following directional domain, signal peptide-linker-scFV-linker-CD 8 alpha hinge-CD 8 alpha transmembrane domain-CD 28-41BB-CD3 zeta-T2A-EG FRt of SEQ ID NO: 4). Self-cleavage of the peptide by T2A allows EGRFt to be expressed on the surface. "suicide" is achieved by cetuximab (anti-EGFR mAb) treatment, which targets T cells for opsonization in vivo.
A second construct, an inducible caspase 9 (iCasp 9) based safety suicide switch, was generated. The iCasp9 molecule is co-expressed on the CAR plasmid following the T2A peptide sequence (the resulting CAR has the amino acid sequence of SEQ ID NO:8 and has the domain of the signal peptide-linker-scFv-linker-CD 8. Alpha. Hinge-CD 8. Alpha. Transmembrane domain-CD 28-41BB-CD3 zeta-T2A-iCasp 9 of SEQ ID NO: 4). Self-cleavage of the peptide by T2A allows iCasp9 to be expressed on the surface. "suicide" is achieved by treatment with AP1903 (rimiducid), AP1903 causing iCasp9 dimerization and triggering the apoptotic pathway.
Transduction efficiency of suicide CAR vectors based on anti-FLT 3 scFv surface expression in human T cells was measured. Expression of scFv was detected using polyclonal anti-Fab APC antibody (Jackson ImmunoResearch, accession number 109-607-003). T cells were stained with scFv detection antibody for 30min at 4 ℃. Cells were washed once with buffer and resuspended in 200 μl of buffer+1:100 7-AAD viability staining solution (Biolegend, accession number 420404) and analyzed by flow cytometry. Flow cytometry acquisitions were performed using Beckman Coulter CytoFLEX (Beckman Coulter) and analysis was performed using FlowJo (TREESTAR INC, ashland, OR). The CAR-T frequencies in anti-FLT 3 CAR (SEQ ID NO: 16), CAR3a-EGFRt (SEQ ID NO: 7) and CAR3a-icasp9 (SEQ ID NO: 8) samples were determined to be 35.3%, 27.5% and 16.9%, respectively (FIG. 9A).
Cytotoxicity assessment of suicide CAR3a constructs CAR3a-EGFRt and CAR3a-icasp9
In vitro cytotoxicity of CAR-T cells with suicide switches CAR3a-EGFRt and CAR3a-icasp9 against AML cell lines compared to the original construct anti-FLT 3 CAR3a was measured. NOMO-1 cells (AML cells expressing FLT 3) were labeled using CELLTRACE TM Violet cell proliferation kit (Thermo FISHER SCIENTIFIC, accession No. C34571). CART cells (effector cells) were combined with labeled NOMO-1 (target cells) at different effector to target cell ratios (10:1, 5:1, 2:1, 1:1, 1:2, and 1:5). The total T cell amount used remained the same regardless of differences in CAR-T frequency (fig. 9B). After 24 hours of co-culture, all cells were collected, labeled with 7-AAD viability staining solution (Biolegend, accession number 420404) and analyzed by flow cytometry. Flow cytometry acquisitions were performed using Beckman Coulter CytoFLEX (Beckman Coulter) and analysis was performed using FlowJo (TREESTAR INC, ashland, OR). Representative dot plots show flow cytometry data after debris removal. Target cells were identified as CellTraceViolet + and effector cells were identified as CellTraceViolet -. The frequency of 7AAD + dying target cells when incubated with T cells expressing CAR-3a, CAR3a-EGFRt and CAR3a-icasp9 at different effective target ratios was measured. All CAR-T cells showed significantly stronger cytotoxic effects on FLT3 + NOMO-1 cells compared to control T cells. There was no significant difference in cytotoxicity effect between the two suicide CAR-T cells (i.e., anti-FLT 3 CAR-EGFRt and anti-FLT 3 CAR-icasp 9) and the original CAR construct (anti-FLT 3 CAR3 a) (fig. 9C).
Example 8: cetuximab is depleted in vitro via ADCC mediated CAR-T as a functional test of EGFRt-CART cell suicide switch
This example demonstrates the successful depletion of CAR T cells expressing the EGFRt suicide gene via antibody dependent cytotoxicity.
To verify expression of the EGFRt suicide switch, human T cells were transduced with a plasmid (SEQ ID NO: 7) expressing the CAR3a-T2A-EGFRt lentiviral vector depicted in FIG. 12B and encoding the domain in the direction of the signal peptide-linker-CD 8 alpha hinge-CD 8 alpha transmembrane domain-CD 28-41BB-CD3 ζ -T2A-EGFRt of scFV-linker-CD 8 alpha hinge-CD 8 alpha transmembrane domain of SEQ ID NO: 4. Surface expression of anti-FLT 3 CAR3a was detected using polyclonal anti-Fab APC antibodies (Jackson ImmunoResearch, numbered 109-607-003). Surface expression of EGFRt was confirmed using cetuximab (SELLECKCHEM A2000) and goat anti-human IGG FC FITC antibody (Jackson) as a secondary antibody (fig. 10A).
An antibody-dependent cellular cytotoxicity (ADCC) assay was performed to measure cetuximab depletion via ADCC-mediated CAR3a-T2A-EGFRt T cells. Specifically, CAR3a-T2A-EGFRt transduced T cells were expanded as described above. On day 8, all or T cell depleted allogeneic Monocytes (MNCs) (from New York Blood Center (NYBC)) (effector cells) were labeled using negative magnetic separation (StemCELL Technologies, no. 17951) using CELLTRACE as previously described and added to transduced T cell (target cell) cultures at a 10:1 ratio of effector cells to target cells. Cetuximab is added to the co-culture at a concentration ranging from 1 to 10000ng/mL. On day 12, all cells from the co-cultures were collected and stained to detect scFv as previously described and analyzed by flow cytometry (fig. 10B). Transduced T cells (no PBMCs) cultured alone showed no significant decrease in CAR3a expressing cells after treatment with cetuximab (fig. 10C). Transduced T cells cultured with total allogeneic MNCs (PBMCs) showed dose-dependent depletion of CAR3a expressing cells by cetuximab. Transduced T cells cultured with T cell depleted allogeneic MNCs (PBMC-T cells) showed dose-dependent depletion of CAR3a expressing cells by cetuximab. The results support the function of Antibody Dependent Cellular Cytotoxicity (ADCC) in vitro on CART cells expressing EGFRt.
Example 9: efficacy of EGFRt-CAR T against AML in vivo and cetuximab in vivo via ADCC-mediated CAR-T depletion
This example demonstrates that anti-FLT 3 CAR T cells expressing the EGFRt suicide gene are able to increase survival in mice with AML (MOLM-13 cells) and are depleted via antibody-dependent cytotoxicity following cetuximab treatment.
Anti-FLT 3 EGFRt-CAR-T cells (as described in example 8) were evaluated for in vivo efficacy against leukemia in female nod.cg-Prkdc scid Il2rgtm1Sug/JicTac (abbreviated NOG mice, taconic, numbered NOG-F hereinafter) transplanted with AML cell line MOLM-13 (DSMZ, numbered ACC 554) transduced to express EGFP. For each NOG mouse (n=15), 2×10 5 EGFP-MOLM-13 cells were transplanted intravenously on day 1. Also on day 1, each mouse received 10×10 6 control T cells (n=5) or 10×10 6 EGFRt-CAR-T cells (20% CAR +) (n=10). On day 18, all mice (n=15) received 9×10 6 effector cells (MNCs depleted of T cells), and one group of EGFRt-CART mice (n=5) also received intraperitoneal injections of 200ug of cetuximab. Three groups of mice were CAR T (-) cetuximab (n=5) (no cetuximab), CAR T (+) cetuximab (n=5), and control T (-) cetuximab (n=5) (fig. 11A). Survival curves were generated up to 65 days after AML injection. Mice exhibiting physiological distress, cachexia or hind leg paralysis were sacrificed. FLT3 CAR-T treatment (induction of no CART suicide) prolonged median survival to 45 days compared to 19 days in control T cell mice. The median survival of CAR t+cetuximab mice was 52 days (fig. 11B). Mice were bled every two weeks to determine AML and T cell engraftment. PBMC were stained with anti-mouse CD45 APC (BioLegend, accession number 103112), anti-human CD45 APC-eFluor780 (Invitrogen, accession number 47045942) and anti-human CD3 PE-Cy7 (BioLegend, accession number 300420) and analyzed by flow cytometry to determine the frequency of MOLM-13 cells (mCD 45 -hCD45+EGFP+) and T cells (mCD 45 -hCD45+CD3+). at week 2 (left), CAR-T cell treated mice showed much less MOLM-13 proliferation than control T cell treated mice. By weeks 4 and 6, all control T cell mice were found to die or euthanized. (right) T cell frequencies (mCD 45 -hCD45+CD3+) in peripheral blood of each group were determined at different time points (fig. 11C). The relative levels of CAR-specific DNA were also quantified by qPCR at weeks 4 and 6, determining the relative amount of circulating CAR-T cells in mice. cetuximab treatment effectively reduced the frequency of circulating CAR-T cells (fig. 11D).
Sequence listing
Incorporated by reference
Various references, such as patents, patent applications, and publications, are cited herein, the disclosures of which are hereby incorporated by reference in their entireties.

Claims (65)

1. A humanized antibody or antigen-binding fragment thereof that binds human FLT3, wherein the antibody or fragment comprises:
i. A light chain variable region (VL) comprising an amino acid sequence ：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37 having at least 95% identity to any one of the sequences selected from the group consisting of SEQ ID NOs 38; and/or
A heavy chain variable region (VH) comprising an amino acid sequence ：SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO;24、SEQ ID NO:25、SEQ ID NO:26 and SEQ ID No. 27 having at least 95% identity to any one of the sequences selected from the group consisting of SEQ ID NOs.
2. The humanized antibody or fragment of claim 1, wherein (i) the VL comprises Complementarity Determining Regions (CDRs) having at least 97%, 98%, 99% or 100% identity to the amino acid sequences of CDR-L1 of SEQ ID No. 86, CDR-L2 of SEQ ID No. 87, and CDR-L3 of SEQ ID No. 88; and (ii) the VH comprises a CDR which has at least 97%, 98%, 99% or 100% identity to the amino acid sequences of CDR-H1 of SEQ ID NO:89, CDR-H2 of SEQ ID NO:90 and CDR-L3 of SEQ ID NO: 91.
3. The humanized antibody or fragment of claim 1, wherein the VL comprises the amino acid sequence of SEQ ID No. 1 and the VH comprises the amino acid sequence of SEQ ID No. 3.
4. The humanized antibody or fragment of claim 1, wherein the VL comprises the amino acid sequence of SEQ ID No. 2 and the VH comprises the amino acid sequence of SEQ ID No. 3.
5. A single chain variable domain (scFv) comprising the antigen binding fragment of any one of claims 1-4.
6. The scFv of claim 5 comprising a linker between the VL and the VH, wherein the linker is of formula (Gly 3-4-Ser)1-4.
7. The scFv of claim 5, wherein the scFv has an amino acid sequence selected from the group consisting of: SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49.
8. The scFv of claim 7, wherein the scFv has the amino acid sequence of SEQ ID No. 4.
9. The scFv of claim 7, wherein the scFv has the amino acid sequence of SEQ ID No. 5.
10. A Chimeric Antigen Receptor (CAR), wherein the CAR comprises: (i) An extracellular domain comprising (a) the antibody or fragment of any one of claims 1-4, or (b) the scFv of any one of claims 5-9; (ii) a transmembrane domain; and (iii) an intracellular domain.
11. The CAR of claim 10, wherein the transmembrane domain is a CD3 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, or a CD28 transmembrane domain.
12. The CAR of claim 10 or 11, wherein the intracellular domain comprises an activation domain, wherein the activation domain transmits an activation signal upon binding of the extracellular domain to FLT3 when the CAR is expressed in a T cell.
13. The CAR of claim 10 or 11, wherein the intracellular domain comprises an activation domain, wherein the activation domain comprises an intracellular signaling domain of cd3ζ, cd3ε, or fcrγ.
14. The CAR of claim 12 or 13, wherein the intracellular domain further comprises one or more co-stimulatory domains.
15. The CAR of claim 14, wherein the one or more co-stimulatory domains is from one or more of: CD28, 4-1BB, CD27, OX40 or ICOS.
16. The CAR of claim 15, wherein the one or more co-stimulatory domains is from CD28 and/or 4-1BB.
17. The CAR of any one of claims 10-16, wherein the CAR comprises a spacer or hinge region between the extracellular domain and the transmembrane domain.
18. The CAR of claim 17, wherein the spacer or hinge region is from the extracellular domain of CD 8.
19. The CAR of any one of claims 10-18, wherein the extracellular domain further comprises a cleavable signal peptide.
20. The CAR of claim 10, wherein the extracellular domain comprises an scFv comprising the amino acid sequence of SEQ ID No. 4; the transmembrane domain comprises a CD8 transmembrane domain; and the intracellular domain comprises an intracellular signaling domain of cd3ζ and a costimulatory domain of CD28 and/or 4-1 BB.
21. The CAR of claim 10, wherein the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15.
22. The CAR of any one of claims 10-20, further comprising a safety switch polypeptide, wherein the safety switch polypeptide binds to the CAR through a self-cleaving peptide.
23. The CAR of claim 22, wherein the safety switch polypeptide is iCasp9 or EGFRt, and wherein the self-cleaving peptide is T2A, P2A, E2A, F a or IRES.
24. The CAR of any one of claims 10-23, wherein when the extracellular domain binds to FLT3, T cells expressing the CAR are activated or stimulated to proliferate.
25. The CAR of any one of claims 10-24, wherein when the CAR is expressed on the surface of a T cell, the CAR directs the T cell to kill FLT3 expressing cells.
26. An immune cell expressing the CAR of any one of claims 10-25 or comprising a nucleic acid encoding the CAR of any one of claims 10-25.
27. The immune cell of claim 26, wherein the immune cell is a T cell, NK cell, macrophage or monocyte.
28. The immune cell of claim 26 or 27, wherein the immune cell is a T cell.
29. The immune cell of any one of claims 26-28, wherein the immune cell comprises a nucleic acid, wherein the nucleic acid comprises a sequence selected from the group consisting of seq id nos: SEQ ID NO. 60, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68 and SEQ ID NO. 69.
30. The immune cell of any one of claims 26-29, wherein the immune cell is derived from a subject prior to introduction of the CAR or the nucleic acid.
31. The immune cell of claim 30, wherein the subject is a human.
32. The immune cell of any one of claims 26-31, wherein the immune cell expressing the CAR or comprising the nucleic acid is further expanded to generate a population of cells.
33. A population of immune cells expressing the CAR of any one of claims 10-25 or comprising a nucleic acid encoding the CAR of any one of claims 10-25.
34. A pharmaceutical composition comprising (i) the humanized antibody or fragment of any one of claims 1-4, the scFv of any one of claims 5-9, the immune cell of any one of claims 26-32, or the population of immune cells of claim 33, and (ii) a pharmaceutically acceptable carrier.
35. A method of treating hematological cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of: (i) The immune cell of any one of claims 26-32, (ii) the population of immune cells of claim 33, or (ii) the pharmaceutical composition of claim 34.
36. The method of claim 35, wherein the hematological cancer is Acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Myeloid Leukemia (CML), chronic Lymphoblastic Leukemia (CLL), blast plasmacytoid dendritic cell tumor (BPDCN), peripheral T cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, non-malignant hereditary or acquired bone marrow disorder, multiple myeloma, or dendritic cell tumor.
37. The method of claim 36, wherein the hematological cancer is AML.
38. The method of claim 36, wherein the hematological cancer is ALL.
39. The method of claim 36, wherein the hematological cancer is a dendritic cell tumor.
40. A method for preparing or conditioning a subject in need thereof for hematopoietic cell transplantation, wherein the method comprises administering to the subject a therapeutically effective amount of: (i) The immune cell of any one of claims 26-32, (ii) the population of immune cells of claim 33, or (ii) the pharmaceutical composition of claim 34.
41. The method of claim 40, wherein the therapeutically effective amount reduces the population of FLT3 expressing cells of the subject by at least 90%.
42. The method of claim 40 or 41, wherein the subject in need thereof has hematological cancer.
43. The method of claim 42, wherein the hematological cancer is Acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Myeloid Leukemia (CML), chronic Lymphoblastic Leukemia (CLL), blast plasmacytoid dendritic cell tumor (BPDCN), peripheral T cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, neuroblastoma, non-malignant hereditary or acquired bone marrow disorders, multiple myeloma, or dendritic cell tumor.
44. The method of claim 43, wherein the hematological cancer is AML.
45. The method of claim 43, wherein the hematological cancer is ALL.
46. The method of claim 43, wherein the hematological cancer is a dendritic cell tumor.
47. The method of any one of claims 35-46, wherein the administration reduces circulating myeloid lineage in the subject, optionally wherein the administration reduces circulating myeloid lineage by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% relative to baseline levels.
48. The method of any one of claims 35-47, wherein the administration reduces bone marrow lineage frequency and number in the subject, optionally wherein the administration reduces bone marrow frequency and/or number by at least 50%, at least 55%, or at least 60% relative to a baseline level.
49. The method of any one of claims 35-48, wherein the administration specifically targets human CD34 + hematopoietic stem cells and/or hematopoietic progenitor cells.
50. The method of claim 49, wherein the administering reduces the population of human CD34 +CD38+ cells in the subject's bone marrow mononuclear cells by at least 50%, at least 55%, at least 60%, or at least 65% relative to a baseline level and/or reduces the population of human CD34 +CD38- cells in the subject's bone marrow mononuclear cells by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% relative to a baseline level.
51. The method of any one of claims 35-50, further comprising performing hematopoietic cell transplantation to the subject following the administering.
52. The method of claim 51, wherein the hematopoietic cell transplantation comprises transplanting hematopoietic stem cells and/or hematopoietic progenitor cells to the subject.
53. The method of claim 51 or 52, wherein said performing of said hematopoietic cell transplantation occurs 5 days to 6 weeks after said administering.
54. The method of claim 53, wherein said performing of said hematopoietic cell transplantation occurs 2 to 3 weeks after said administering.
55. The method of any one of claims 35-54, wherein the therapeutically effective amount of the immune cell or population of immune cells is a dose of about 50,000,000 to 10,000,000,000 cells.
56. The method of claim 55, wherein the therapeutically effective amount of the immune cell or population of immune cells is a dose of about 100,000,000 to 2,000,000,000 cells.
57. The method of any one of claims 35-56, wherein the administration is intravenous.
58. The method of claim 57, wherein the intravenous administration is by infusion into the subject.
59. The method of any one of claims 35-58, wherein the administration occurs once.
60. The method of any one of claims 35-58, wherein the administration is once every 3-7 days for 2 to 3 weeks.
61. The method of any one of claims 35-60, wherein the method comprises the following steps prior to the administering step:
(v) Collecting blood from the subject;
(vi) Isolating immune cells from the blood;
(vii) Introducing into the isolated immune cell a nucleic acid encoding the CAR of any one of claims 10-25; and
(Viii) Amplifying the isolated immune cells obtained in step (iii), wherein the amplifying produces the immune cells or the population of immune cells administered during the administering step.
62. The method of any one of claims 35-61, further comprising administering a checkpoint inhibitor.
63. The method of claim 62, wherein the checkpoint inhibitor is an antagonist of PD1, PD-L1 or CTLA 4.
64. The method of claim 63, wherein the antagonist is an antagonistic antibody.
65. The method of any one of claims 35-64, wherein the subject is a human.
CN202280068117.6A 2021-08-16 2022-08-15 Anti-FLT 3 antibodies, CARs, CAR T cells, and methods of use Pending CN118284621A (en)

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