EA044866B1 - CHIMERIC RECEPTORS FOR FLT3 AND METHODS OF THEIR APPLICATION - Google Patents

Description

Prerequisites for creating the invention

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that is the most common type of acute leukemia diagnosed in adult patients. AML accounts for approximately a third of all leukemia cases, given the expected 14,500 new cases reported in 2013 in the US alone and the low overall survival rates. There has been little improvement in the standard of care for patients with AML over the past thirty years. However, recent advances in molecular and cellular biology have radically changed our understanding of human hematopoiesis, both in health and disease. Several key players involved in disease pathogenesis have been identified and can be targeted in detail. One of these activating driver genes that is most commonly mutated in approximately 30% of AML is FLT3.

Fms-like tyrosine kinase 3 (FLT3), also known as germline liver kinase 2 (FLK-2), human stem cell kinase 1 (SCK-1), or cluster of differentiation antigen (CD135), is a hematopoiesis-associated receptor tyrosine kinase that is In the 1990s, two independent groups cloned. The FLT3 gene, located on chromosome 13q12 in humans, encodes the class III receptor tyrosine kinase protein, which shares homology with other members of the class III family, including stem cell factor receptor (c-KIT), macrophage colony-stimulating factor (FMS) receptor. and platelet-derived growth factor receptor (PDGFR).

Upon binding to FLT3 ligand, the FLT3 receptor undergoes homogenization, thereby allowing autophosphorylation of specific tyrosine residues in the juxtamembrane domain and subsequent activation through the PI3K/Akt, MAPK, and STAT5 pathways. Thus, FLT3 plays a critical role in the control of proliferation, survival, and differentiation of normal hematopoietic cells. Human FLT3 is expressed in CD34+CD38- hematopoietic stem cells (HSCs) as well as a subpopulation of dendritic progenitor cells. FLT3 expression can also be found in multipotent progenitor cells, such as conventional myeloid progenitor cells (CMP) CD34 ⁺ CD38 ⁺ CD45RA-CD123 ^low , granulocyte and monocyte progenitor (GMP) cells CD34 ⁺ CD38 ⁺ CD45RA ⁺ CD123 ^low , and general lymphoid progenitor cells (CLP) CD34 ⁺ CD38 ⁺ CD10 ⁺ CD19-. Notably, FLT3 expression is virtually absent in CD34 ⁺ CD38'CD45RA'CD123' megakaryocyte and erythrocyte progenitor (MEP) cells. Thus, FLT3 expression is primarily found in early myeloid and lymphoid progenitor cells, with some expression in more mature monocytes. This limited expression profile of FLT3 contrasts strikingly with the expression profile of FLT3 ligand, which is expressed in most hematopoietic tissues and in the prostate, kidney, lung, colon and heart. These different expression profiles, such as FLT3 expression, represent a rate-limiting step in determining the tissue specificity of FLT3 signaling pathways.

The most common FLT3 mutation in AML is FLT3 internal tandem duplication (FLT3-ITD), which is found in 20-38% of patients with cytogenetically normal AML. FLT3-ITDs are formed when part of a juxtamembrane domain coding sequence is duplicated and inserted in a head-to-tail orientation. FLT3 mutations have not been identified in patients with chronic lymphoid leukemia (CLL), non-Hodgkin's lymphoma, and multiple myeloma, suggesting strong disease specificity for AML. Mutant FLT3 activation is commonly observed in all FAB subtypes, however, it is significantly increased in AML patients with FAB M5 (monocytic leukemia), whereas FAB subtypes M2 and M6 (granulocytic or erythroid leukemia) are significantly less likely to be associated with FLT3 activation , in a line with normal FLT3 expression profiles. A small percentage of AML patients (5-7%) have single amino acid mutations in the FLT3 tyrosine kinase domain (FLT3 TKD), most commonly D835 or, in some cases, T842 or 1836, while an even smaller number of patients (~1 %) have a cryptic mutation in the juxtamembrane domain of FLT3 involving residues 579, 590, 591, and 594. Patients with mutated FLT3-ITD AML have an aggressive form of the disease that is characterized by early relapse and poor survival, whereas overall survival and event-free survival are not significantly affected influence of the presence of FLT3-TKD mutations. In addition, patients with FLT3-ITD mutated AML with concurrent TET2 or DNMT3A mutations have an undesirable overall risk profile compared with patients with FLT3-ITD mutated AML with wild-type TET2 or DNMT3A, highlighting the clinical and biological heterogeneity AML.

Both FLT3-ITD and FLT3 TKD mutations involve ligand-dependent activation of FLT3, which leads to subsequent activation of the Ras/MAPK pathway and PI3K/Akt pathways. However, the downstream signaling pathways associated with any mutation differ primarily in that STAT5 is preferentially activated by FLT3-ITD, thereby leading to increased proliferation potential and misregulation of DNA repair pathways.

Regardless of FLT3 mutation status, FLT3 phosphorylation occurs in more than two

- 1,044,866 of AML patients, and FLT3 is expressed in more than 80% of AML blasts and in approximately 90% of all AML patients, making it an attractive therapeutic target associated with disease pathogenesis in large sample sizes.

Several small molecule inhibitors have emerged as attractive potential treatment options for AML patients with FLT3 mutations. The first generation of FLT3 tyrosine kinase inhibitors (TKIs) were characterized by a lack of selectivity, potency, and undesirable pharmacokinetic properties. New and more selective agents have never been developed to combat this issue; however, their effectiveness has been limited by the emergence of secondary resistance. Several early FLT3 TKIs included midostaurin (PKC412), lestaurtinib (CEP-701), sunitinib (SUI1248), and sorafinib (BAY 43-9006), among others. Response rates in phase I and phase II with these multi-kinase targeting agents in patients with relapsed or refractory AML are limited, presumably due to their inability to achieve effective FLT3 inhibition without dose-limiting toxicities. Quizartinib (AC220) was developed as a second-generation FLT3 TKI with high selectivity for wild-type and FLT3-ITD FLT3, and has demonstrated benefit particularly in perigraft settings in a younger patient population. However, secondary mutations in FLT3 found in relapsed patients treated with quizartinib highlight the need to develop better therapeutic strategies for patients with AML, while highlighting the validity of FLT3 as a therapeutic target.

Several targeted agents have been tested in AML patients with either primary, relapsed/refractory, or secondary disease. Epigenetic silencing of tumor suppressor genes plays an important role in the pathogenesis of AML disease, and DNA methyltransferase (DNMT) inhibitors such as azacitadine and decitabine have achieved some clinical success. In addition, the recent discovery of mutations that negatively impact post-translational histone modifications (eg, EZH2 and ASXL1 mutations) or DNA methylation (eg, DNMT3A, TET2, IDH1/2) in a subpopulation of AML patients has provided the development of a variety of potential treatments, including including EZH2, DOT1L, IDH1/2 inhibitors along with HDAC and protease inhibitors. However, preclinical studies of a variety of these components in AML cells suggest that these inhibitors may alter the phenotypic and gene expression characteristics of hematopoietic differentiation rather than cause direct cytotoxicity of AML blasts. Consequently, there remains a strong unmet medical need to identify new targets/methods to combat AML and induce targeted lysis of AML blast cells. Other therapeutic candidates for AML include Aurora kinase inhibitors, including AMG 900, and inhibitors for Polo-like kinases, which play important roles in cell cycle progression. The standard of care for patients with AML continued to be chemotherapy with stem cell transplantation when possible. However, the occurrence of relapse/resistance in a large majority of treated patients warrants additional therapeutic options. The identification and characterization of several leukemia-specific antigens, together with a clearer understanding of the immune-mediated graft-versus-leukemia effects, have paved the way for the development of immunomodulatory strategies to combat hematologic malignancies, reviewed in several articles. The engineered immune cells have been shown to have the necessary qualities in therapeutic treatments, particularly in oncology. The two main types of immune cells developed are cells that contain chimeric antigen receptors (called CARs or CAR-Ts) and T cell receptors (TCRs). These engineered cells are designed to be antigen specific to them, maintaining or enhancing their ability to recognize and kill a target cell. Chimeric antigen receptors may contain, for example, (i) an antigen-specific component (an antigen-binding molecule), (ii) one or more co-stimulatory domains, and (iii) one or more activating domains. Each domain can be heterogeneous, that is, it can consist of sequences formed from different protein chains. Chimeric antigen receptor-expressing immune cells (eg, T cells) can be used in a variety of therapies, including cancer therapies. It should be understood that costimulatory polypeptides, as defined herein, can be used to enhance the activation of CAR-expressing cells against target antigens and, therefore, increase the potency of adoptive immunotherapy.

T cells can be engineered to have specificity for one or more desired targets. For example, T cells can be transfected with DNA or other genetic material encoding an antigen-binding molecule, such as one or more single chain antibody variable fragments (scFv), together with one or more signaling molecules, and/or one or more activating domains, for example CD3 zeta.

In addition to the ability of CAR-T cells to recognize and destroy target cells, successful T-cell therapy enhances the ability of CAR-T cells to persist and maintain the ability to proliferate in response to antigen.

T cell receptors (TCRs) are molecules found on the surface of T cells that

- 2 044866 which are responsible for recognizing antigen fragments as peptides associated with the main molecules of the histocompatibility complex (MHC). The TCR is composed of two different protein chains. In approximately 95% of human TCRs, the TCR is composed of an alpha (α) and beta (β) chain. In approximately 5% of human T cells, the TCR consists of gamma and delta (γ/δ) chains, each chain composed of two extracellular domains: a variable region (V) and a constant region (C), both from the immunoglobulin superfamily. As with other immunoglobulins, the TCR α chain and β chain variable domains (or gamma and delta (γ/δ) chains) each have three hypervariable or complementarity determining regions (CDRs). If the TCR interacts with an antigenic peptide and MHC (peptide/MHC), the T cell becomes activated, causing it to defeat and kill the target cell.

However, currently available treatment options have shown variable levels of effectiveness with unwanted side effects. Therefore, there is a need to identify new and improved therapeutic options for the treatment of FLT3-related diseases and disorders.

Summary of the invention

The present invention relates to engineered immune cells (eg, CAR or TCR), antigen binding molecules (including, without limitation, antibodies, scFvs, heavy and/or light chains and CDRs of these antigen binding molecules) with specificity for FLT3.

The present invention also provides a new CD28 sequence useful as co-stimulatory domains in these cells.

The chimeric antigen receptors of the present invention generally include the following: (i) a FLT3-specific antigen binding molecule, (ii) one or more costimulatory domains, and (iii) one or more activating domains. It should be understood that each domain may be heterogeneous, thus consisting of sequences derived from different protein chains.

In some embodiments, the present invention provides a chimeric antigen receptor comprising an antigen binding molecule that specifically binds to FLT3, wherein the antigen binding molecule comprises at least one of the following: (a) a heavy chain CDR1 variable region comprising an amino acid sequence that differs from sequences in SEQ ID NO: 17 of no more than 3, 2, 1 or 0 amino acid residues; (b) a heavy chain CDR2 variable region containing an amino acid sequence that differs from the sequence in SEQ ID NO: 18 or SEQ ID NO: 26 by no more than 3, 2, 1 or 0 amino acid residues; (c) a heavy chain CDR3 variable region containing an amino acid sequence that differs from the sequence in SEQ ID NO: 19 or SEQ ID NO: 27 by no more than 3, 2, 1 or 0 amino acid residues; (d) a light chain CDR1 variable region containing an amino acid sequence that differs from the sequence in SEQ ID NO: 22 or SEQ ID NO: 30 by no more than 3, 2, 1 or 0 amino acid residues; (f) a light chain CDR2 variable region containing an amino acid sequence that differs from the sequence in SEQ ID NO: 23 or 31 by no more than 3, 2, 1 or 0 amino acid residues; (f) a light chain CDR3 variable region containing an amino acid sequence that differs from the sequence in SEQ ID NO: 24 or SEQ ID NO: 32 by no more than 3, 2, 1, or 0 amino acid residues.

In other embodiments, the chimeric antigen receptor further comprises at least one costimulatory domain. In further embodiments, the chimeric antigen receptor further comprises at least one activating domain.

In some embodiments, the costimulatory domain is CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cell costimulator signaling region ( ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll receptor -ligands, ICAM-1, B7H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta , IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA1, ITGAM, CDl lb, ITGAX, CDl lc, 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, a ligand that specifically binds CD83, or any combination thereof.

In some embodiments, the co-stimulatory domain is derived from 4-1BB. In other embodiments, the co-stimulatory domain is derived from OX40. See also Hombach et al., Oncoimmu

- 3 044866 nology. 2012 Jul. 1; 1(4): 458-466. In further embodiments, the co-stimulatory domain comprises ICOS, as described in Guedan et al., August 14, 2014; Blood: 124 (7) and Shen et al., Journal of Hematology & Oncology (2013) 6:33. In further embodiments, the costimulatory domain comprises CD27, as described in Song et al., Oncoimmunology. 2012 Jul. 1; 1(4):547-549.

In some embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In further embodiments, the CD8 costimulatory domain comprises SEQ ID NO: 14. In further embodiments, the activating domain contains CD3, CD3 zeta, or CD3 zeta with the sequence shown in SEQ ID NO: 10.

In other embodiments, the present invention provides a chimeric antigen receptor wherein the co-stimulatory domain comprises SEQ ID NO: 2 and the activating domain comprises SEQ ID NO: 10.

The present invention also relates to polynucleotides encoding chimeric antigen receptors and vectors containing the polynucleotides. The vector may be, for example, a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-related vector, a lentiviral vector, or any combination thereof. The present invention also relates to immune cells containing vectors. In some embodiments, the lentiviral vector is a pGAR vector.

Exemplary immune cells include, but are not limited to, T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK T cells. T cells can be autologous, allogeneic or heterologous. In other embodiments, the present invention relates to pharmaceutical compositions containing immune cells, as described herein.

In some embodiments, the present invention provides antigen-binding molecules (and chimeric antigen receptors containing these molecules) containing at least one of the following:

(a) a VH region that differs from the amino acid sequence of the VH region in 10E3 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region in 10E3 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(b) a VH region that differs from the amino acid sequence of the VH region in 2E7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region in 2E7 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(c) a VH region that differs from the amino acid sequence of the VH region in 8B5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region in 8B5 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(d) a VH region that differs from the amino acid sequence of the VH region in 4E9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region in 4E9 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

(e) a VH region that differs from the amino acid sequence of the VH region in 11F11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues, and a VL region that differs from the amino acid sequence of the VL region in 10E3 of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acid residues;

and wherein the VH and VL region or regions are linked by at least one linker.

In other embodiments, the present invention provides antigen binding molecules (and chimeric antigen receptors comprising these molecules), wherein the linker comprises at least one of a G4S scFv linker and a Whitlow scFv linker.

In other embodiments, the present invention relates to vectors encoding the polypeptides of the present invention, and to immune cells containing these polypeptides. Preferred immune cells include T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR expressing cells, dendritic cells or NK T cells. T cells can be autologous, allogeneic, or heterologous.

In other embodiments, the present invention provides isolated polynucleotides encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) comprising an antigen binding molecule that specifically binds to FLT3, wherein the antigen binding molecule comprises a heavy chain CDR3 variable region ( _VH ) containing the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 27. The polynucleotides may further comprise an activating domain. In preferred embodiments, the activating domain is CD3, more preferably CD3 zeta, more preferably the amino acid sequence set forth in SEQ ID NO: 9.

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In other embodiments, the present invention includes a co-stimulatory domain, for example CD28, CD28T, OX40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CDl la/CD18) , CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signaling lymphocyte activation molecule, 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, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld , ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-lc, 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, CD83 ligand, or fragments or combinations thereof. Preferred costimulatory domains are presented below in this document.

In further embodiments, the present invention provides isolated polynucleotides encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR), wherein the CAR or TCR comprises an antigen binding molecule that specifically binds to FLT3, and where the antigen binding molecule comprises a CDR3 variable region light chain (V _L ) containing an amino acid sequence selected from SEQ ID NO: 24 and SEQ ID NO: 32. The polynucleotide may further comprise an activating domain. The polynucleotide may further comprise a co-stimulatory domain.

In other embodiments, the present invention provides isolated polynucleotides encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) containing antigen binding molecule that specifically binds to FLT3, wherein the heavy chain of the antigen binding molecule comprises CDR1 (SEQ ID NO: 17 ), CDR2 (SEQ ID NO: 18), and CDR3 (SEQ ID NO: 19) and the light chain of the antigen binding molecule contains CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO : 24).

In other embodiments, the present invention provides isolated polynucleotides encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) containing antigen binding molecule that specifically binds to FLT3, wherein the heavy chain of the antigen binding molecule comprises CDR1 (SEQ ID NO: 17 ), CDR2 (SEQ ID NO: 26), and CDR3 (SEQ ID NO: 27) and the light chain of the antigen binding molecule contains CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO : 32).

The present invention further provides antigen binding molecules that bind to FLT3 comprising at least one heavy chain CDR3 variable region sequence or a light chain CDR3 variable region sequence as defined herein. The present invention further relates to FLT3-binding antigen-binding molecules comprising at least one heavy chain variable region sequence CDR1, CDR2 and CDR3 as described herein. The present invention further relates to FLT3-binding antigen-binding molecules comprising at least one light chain variable region sequence CDR1, CDR2 and CDR3, as described herein. The present invention further provides antigen binding molecules that bind to FLT3 containing both heavy chain variable region sequences CDR1, CDR2, CDR3 and light chain variable region sequences CDR1, CDR2 and CDR3, as described herein.

Additional heavy and light chain variable domains, CDR polynucleotide and amino acid sequences useful for use in FLT3 binding molecules in accordance with the present invention are found in Provisional Patent Application No. 62/199944, filed July 31, 2015.

The present invention further provides methods for treating a disease or disorder in a subject in need thereof, comprising administering to the subject antigen binding molecules, CARs, TCRs, polynucleotides, vectors, cells or compositions of the present invention. Suitable diseases for treatment include, but are not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma) or combinations thereof. Additional diseases include inflammatory and/or autoimmune diseases, such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and celiac disease.

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Brief description of graphic materials

In fig. Figure 1 shows flow cytometry of cell surface expression of FLT3 in human cell lines.

In fig. 2 shows CAR expression in primary human T cells electroporated with mRNA encoding various CARs.

In fig. Figure 3 shows the cytolytic activity of electroporated CAR T cells compared to several cell lines after 16 hours of coculture.

In fig. 4, including FIG. 3A and 3B show the production of IFNy, IL-2 and TNFa by electroporated CAR T cells after 16 hours of co-culture with the indicated target cell lines.

In fig. Figure 5 shows CAR expression in lentivirus-transfected primary human T cells from two healthy donors.

In fig. 6 shows the average cytolytic activity over time from two healthy donors expressing the indicated CARs co-cultured with different target cell lines.

In fig. 7, including FIG. 7A, 7B and 7C show the production of IFNy, TNFa and IL-2 by lentivirus-transfected human CAR T cells from two healthy donors after 16 hours of co-culture with the indicated target cell lines.

In fig. 8 shows the proliferation of CFSE-labeled lentivirus-transfected CAR T cells from two healthy donors after 5 days of coculture with CD3-CD28 beads or the indicated target cell lines.

In fig. 9 shows CAR expression in lentivirus-transfected primary human T cells suitable for in vivo studies.

In fig. Figure 10 shows bioluminescent imaging of labeled acute myeloid leukemia cells following intravenous injection of CAR T cells in a xenogeneic model.

In fig. 11 shows the survival curves of mice injected with CAR T cells.

In fig. Figure 12 shows the vector diagram of pGAR.

Detailed Description of the Invention

It should be understood that chimeric antigen receptors (CAR or CAR-T) and T-cell receptors (TCR) are genetically engineered receptors. These designed receptors can be easily internalized and expressed by immune cells, including T cells, according to techniques known in the art. With the help of a CAR, one receptor can be programmed to both recognize a specific antigen and, upon binding to that antigen, activate an immune cell to attack and destroy the cell bearing that antigen. If these antigens exist on tumor cells, then the immune cell that expresses the CAR can target and destroy the tumor cell.

CARs can be designed to bind to an antigen (eg, a cell surface antigen) by introducing an antigen-binding molecule that interacts with the target antigen. Preferably, the antigen binding molecule is an antibody fragment thereof and, more preferably, one or more single chain antibody fragments (scFv). scFv is a single chain antibody fragment having variable regions of the antibody heavy and light chains linked together. See US Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. scFv retains the ability of the parent antibody to specifically interact with the target antigen. scFvs are preferred for use in chimeric antigen receptors because they can be designed to be expressed as part of a single chain along with other CAR components. Id. See also Krause et al., J. Exp. Med., ed. 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797. It should be understood that the antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the present invention with specificity for more than one target of interest.

Costimulatory domains

Chimeric antigen receptors can introduce costimulatory (signaling) domains to increase their potency. See US Patent Nos. 7,741,465 and 6,319,494 and Krause et al. and Finney et al. (above), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). For example, CD28 is a co-stimulatory protein found naturally on T cells. The complete original amino acid sequence of CD28 is described in the NCBI exemplary sequence: NP_006130.1. The complete original CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.

Specific domains of CD28 have been used in chimeric antigen receptors. In accordance with

- 6 044866 with the present invention, it has now been discovered that a new extracellular domain of CD28, called CD28T, unexpectedly provides certain advantages when used in a CAR construct.

The nucleotide sequence of the CD28T molecule, including the extracellular domain of CD28T, and the transmembrane and intracellular domains of CD28 are listed in SEQ ID NO: 1:

CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAA

GCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGTTGGTC

GTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGTCGTCACCGTGGCTTTTATAATCT

TCTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTC

CACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGAT

TTCGCTGCCTATCGGAGC

The corresponding amino acid sequence is listed in SEQ ID NO: 2:

LDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTV

AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRS

The nucleotide sequence of the extracellular portion of CD28T is listed in SEQIDNO: 3:

CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAA

GCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA

The corresponding amino acid sequence of the extracellular domain of CD28T is listed in SEQ ID NO: 4:

LDNEKSNGTIIHVKGKHLCP SPLFPGPSKP

The nucleotide sequence of the transmembrane domain of CD28 is listed in SEQ ID NO: 5:

TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGC

TCGTCACCGTGGCTTTTATAATCTTCTGGGTT

The amino acid sequence of the transmembrane domain of CD28 is listed in SEQ ID NO: 6: FWVLVVVGGV LACYSLLVTV AFIIFWV

The nucleotide sequence of the intracellular signaling domain of CD28 is listed in SEQ ID NO: 7:

AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACT

CCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGGCACCACCTAGAGA

TTTCGCTGCCTATCGGAGC

The amino acid sequence of the intracellular signaling domain of CD28 is listed in SEQ ID NO: 8:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

Additional CD28 sequences useful for use in the present invention include the CD28 nucleotide sequence listed in SEQ ID NO: 11:

ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAAC

GGTACCATCATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCCTCTTCCCCGGG

CCATCAAAAGCCC

The corresponding amino acid sequence is listed in SEQ ID NO: 12: IEVMYPPPYLDNEI<SNGTIIHVI<GI<HLCP SPLFPGPSKP

Other suitable extracellular or transmembrane sequences may be derived from CD8. The nucleotide sequence of a suitable extracellular and transmembrane domain of CD8 is listed in SEQ ID NO: 13:

GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCA

GTGTTCTTGCCGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCT

CCTACCATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCA GGGGGCGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCA CCCCTGGCCGGAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGT AATCACAGGAAC

The corresponding amino acid sequence is listed in SEQ ID NO: 14: AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP

AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN

Suitable co-stimulatory domains within the scope of the present invention can be obtained from, among other sources, CD28, CD28T, OX40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1 (CDl la/CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, lymphocyte activation signaling molecule, BTLA, Toll ligand receptor, ICAM-1, B7H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,

- 7 044866

CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA1, ITGAM, CDl-lb, ITGAX, CDl-lc, 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, CD83 ligand, or fragments or combinations thereof.

Activating domains

CD3 is a T cell receptor element on the parent T cell and has been shown to be an important intracellular activating element in CAR. In a preferred embodiment, CD3 is CD3 zeta, the nucleotide sequence of which is listed in SEQ ID NO: 9:

AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGC

CAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTT GGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAAC CCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTC TGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGTTTGTAC CAGGGACTCAGCACTGCTA CGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTG CCACCTAGG

The corresponding intracellular CD3 amino acid zeta is listed in SEQ ID NO: 10: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK

PR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR

Domain orientation

It should be understood that structurally these domains correspond to locations corresponding to immune cells. Thus, these domains may be part of (i) a hinge or extracellular (EC) domain (EC), (ii) a transmembrane (TM) domain, and/or (iii) an intracellular (cytoplasmic) domain (IC). The intracellular component often contains part of a CD3 family member, preferably CD3 zeta, which is capable of activating the T cell upon binding of an antigen binding molecule to its target. In one embodiment, the hinge domain typically consists of at least one co-stimulatory domain, as defined herein.

It should also be understood that the hinge region may also contain some or all members of the immunoglobulin family, for example, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof.

Exemplary CAR designs in accordance with the present invention are listed in table. 1.

_____________________Table 1_____________________

Design name scFv Costimulatory domain Activating domain 24С1 CD28T 24С1 CD28T CD3 zeta 24С1 CD28 24С1 CD28 CD3 zeta 24С1 CD8 24С1 CD8 CD3 zeta 24С8 CD28T 24С8 CD28T CD3 zeta 24С8 CD28 24С8 CD28 CD3 zeta 24С8 CD8 24С8 CD8 CD3 zeta 20С5.1 CD28T 20С5.1 CD28T CD3 zeta 20С5.1 CD28 20С5.1 CD28 CD3 zeta 20С5.1 CD8 20С5.1 CD8 CD3 zeta 20С5.2 CD28T 20С5.2 CD28T CD3 zeta 20С5.2 CD28 20С5.2 CD28 CD3 zeta 20С5.2 CD8 20С5.2 CD8 CD3 zeta

Cell related domains

It should be understood that, with respect to the cell bearing the receptor, the designed T cells of the present invention contain an antigen binding molecule (eg, scFv), an extracellular domain (which may contain a hinge domain), a transmembrane domain, and an intracellular domain. The intracellular domain contains at least in part an activating domain, preferably consisting of members of the CD3 family, for example CD3 zeta, CD3 epsilon, CD3 gamma or parts thereof. Should

- 8 044866 also understand that the antigen binding molecule (eg, one or more scFv) is designed such that it is located in the extracellular portion of the molecule/construct so that it is capable of recognizing and binding to its target or targets.

Extracellular domain

The extracellular domain is positive for signaling and for the effective response of lymphocytes to antigen. Extracellular domains for specific use according to the present invention may be formed from (i.e., consist of) CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD -1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, lymphocyte activation signaling molecules (SLAM proteins), activating 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, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld , ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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, a ligand that specifically binds to CD83, or any combinations thereof. The extracellular domain can be formed from either a natural or synthetic source.

As described herein, extracellular domains often contain a hinge region. It is part of the extracellular domain, sometimes called the spacer region. In accordance with the present invention, a variety of hinges can be used, including co-stimulatory molecules as discussed above, as well as immunoglobulin (Ig) sequences or other suitable molecules, to achieve the desired spatial distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises CD28T or a CD28 EC domain.

Transmembrane domain

The CAR can be designed to include a transmembrane domain that is fused to an extracellular domain of the CAR. It may similarly be condensed to the intracellular CAR domain. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the CAR. In some cases, the transmembrane domain may be selected from or modified by amino acid substitution to avoid binding of such domains to their transmembrane domains or various surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain can be obtained from either a natural or synthetic source. If the source is natural, the domain can be formed from any membrane-associated or transmembrane protein. Transmembrane regions for specific use according to the present invention can be formed from (i.e., consist of) CD28, CD28T, OX-40, 41BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1 ), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14 ), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating 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, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE , CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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, a ligand that specifically binds CD83, or any combination thereof.

Optionally, short linkers can form connections between any or some of the extracellular, transmembrane and intracellular domains of the CAR.

In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a transmembrane portion of the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence that encodes a transmembrane amino acid sequence comprising

- 9 044866 found in SEQ ID NO: 14.

In some embodiments, the transmembrane domain in the CAR of the present invention is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 5. In one embodiment, the CD28 transmembrane domain comprises a nucleic acid sequence that encodes the amino acid sequence in SEQ ID NO: 6. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence sequence in SEQ ID NO: 6.

Intracellular (cytoplasmic) domain

The intracellular (cytoplasmic) domain of the engineered T cells of the present invention may enable activation of at least one of the normal effector functions of the immune cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines.

It should be understood that suitable intracellular molecules include (i.e. contain) but are not limited to CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1) , inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, ( TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK receptors cells, 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, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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, SLP76, PAG/Cbp, CD19a, a ligand that specifically binds CD83, or any combination thereof.

In a preferred embodiment, the cytoplasmic domain of the CAR may be designed to include a CD3 zeta signaling domain, alone or coupled to any other necessary cytoplasmic domain(s) useful in the context of the CARs of the present invention. For example, the cytoplasmic domain of a CAR may contain part of the CD3 zeta chain and a co-stimulatory signaling region.

The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR of the present invention may be linked together in a random or specific order.

In one preferred embodiment, the cytoplasmic domain is designed to include a CD3 zeta signaling domain and a CD28 signaling domain. In another embodiment, the cytoplasmic domain is designed to include a CD3 zeta signaling domain and a 4-1BB signaling domain. In another embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include portions of CD28 and CD3 zeta, wherein the cytoplasmic CD28 comprises the nucleic acid sequence listed in SEQ ID NO: 7 and the amino acid sequence listed in SEQ ID NO: 8. The CD3 zeta nucleic acid sequence is listed in SEQ ID NO: 9, and the amino acid sequence is listed in SEQ ID NO: 8.

It should be understood that one preferred orientation of the CAR in accordance with the present invention contains an antigen binding domain (eg, scFv) together with a costimulatory domain and an activating domain. The costimulatory domain may comprise one or more extracellular portions, a transmembrane portion, and an intracellular portion. It should also be understood that multiple co-stimulatory domains may be used together.

In some embodiments, nucleic acids are provided comprising a promoter operably linked to a first polynucleotide encoding an antigen binding molecule, at least one co-stimulatory molecule, and an activating domain.

In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of retroviral vectors, murine leukemia virus vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, and vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid. The present invention further relates to isolated polynucleotides encoding chimeric antigen receptors and vectors containing the polynucleotides. Any vector known in the art may be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (e.g., pMSVG1), a DNA vector, a leukemia virus vector

- 10 044866 mouse mii, SFG vector, plasmid, RNA vector, adenoviral vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adeno-associated virus (AAV) vector, lentiviral vector ( for example, pGAR) or any combination thereof. The pGAR vector map is shown in Fig. 12. The sequence of pGAR is as follows:

CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGCAGC GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGG GTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAACTTGATTAGGGTGATGG TTCACGTA GTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTC CACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTC GGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT GAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATT TGCCATTCGCCATTCAGGCTGCGCA ACTGTTGGGAAGGGCGATCGGTGCGGGCCTCT TCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGT AACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAAT ACGACTCACTATAGGGCGACCCGGGGATGGCGCGCCAGTAATCAATTACGGGTCA TTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC ATAACTTACGGTAAAATGGCCCG CCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCC ATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAA ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCTGATGCGGT TTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTC TCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTC CAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGG TGGGA GGTCTATATAAGCAGAGCTGGTTTAGTGAACCGGGGTCTCTCTGGTTAGACC AGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAAT AAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCC GAACAGGGACTTGAAA GCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACT CGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCC AAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGATGGGTGCGAGAGCGTCAGT ATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAATATAAATTAAAACATATAGTATGGGCAAGCA GGGAGCTAGAACG ATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGG GACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAAT ACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGA AGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAA GCCGCCGCTGATC TTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTG AATTATATAAATATAAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAG GCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGT TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG ACGGTACAGGCCAGACAATTATTGTCTG GTATAGTGCAGCAGCAGAACAATTTGCT GAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGC AGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTG GGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCT AGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACA T AGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAG

- 11 044866

GGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGA CAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAA AGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACA GACATACAAACTAAAGAATTACAAAAACAAATTACAAAATTCAAAATTTTATCGCG ATCGCGGAATGAAAGACCCCACCT GTAGGTTTGGCAAGCTAGCTTAAGTAACGCCA TTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTA GGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCC TGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCA GTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGA CCTGAAAATGACCCT GTGCCTTATTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGC TCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTTCGAA GTAGATCTTTGTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCTGCCAA GCTTCCGAGCTCTCGAATTAATTCACGGTACCCACCATGGCCTAGGGAGACTAGTC G AATCGATATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAAC TATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAATCCTGGTTGCTGTCTCTT TATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT A CGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTCCGCGGCCTGCT GCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCC CTTTGGGCCGCCTCCCCGCCTGGTTAATTAAAGTACCTTTAAGACCAATGACTTACA AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCGA ATTCACTCCCAACGAAGACAAGATCTG CTTTTTGCTTGTACTGGGTCTCTCTGGTTAG ACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTC AATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTG GTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGCATG CCAGACATGATAAGATACATTGATGAGTTTGGACAAAC CACAACTAGAATGCAGTG AAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATA AGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG GGGGAGGTGTGGGAGGTTTTTTGGCGCGCCATCGTCGAGGTTCCCTTTAGTGAGGGT TAATTGCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTT ATCC GCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG

- 12 044866

CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGGC GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA GAATCA GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTG CCGCTTACCGGATACCTGTCC GCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCT ACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAA ACTCACGTTAA GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAC CAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCC CC AGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCC TCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATG GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC ATGTTGTGCAAAAAAGCGGTTAGCTTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG TTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG AATAGTGTATGCGGCGA CCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC GGAAATGTTGAATAC TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGTTATTGTCTCATGAG CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATT TCCCCGAAAAGTGCCAC (SEQ ID NO: 95)

Additional suitable exemplary vectors include, for example, pBABE-puro, pBABEneo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp- eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXINLuc.

In some embodiments, the engineered immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR expressing cell, a dendritic cell, or an NK T cell. In some embodiments, the cell is obtained or prepared from peripheral blood. In some embodiments, the cell is obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is obtained or prepared from bone marrow. In some embodiments, the cell is obtained or prepared from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, transfected or transformed with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (eg, gene gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes.

In some embodiments, the chimeric antigen receptors are expressed in engineered immune cells that contain the nucleic acids of the present application. These chimeric antigen receptors of the present application, in some embodiments, may contain

- 13 044866 to harvest (i) an antigen binding molecule (eg scFv), (ii) a transmembrane region and (iii) a T cell activating molecule or region.

Antigen-binding molecules

Antigen binding molecules are included within the scope of the present invention.

As used herein, an antigen-binding molecule means any protein that binds to a specified target antigen. In the present application, said target antigen is the FLT3 protein or a fragment thereof. Antigen-binding molecules include, but are not limited to, antibodies and binding portions thereof, such as immunologically functional fragments. Peptide antibodies (ie, Fc condensed molecules containing peptide binding domains) are other examples of useful antigen binding molecules.

In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen. In some embodiments, the antigen binding molecule binds to FLT3. In further embodiments, the antigen binding molecule is an antibody or fragment thereof, including one or more complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain fragment variable (scFv).

The term immunologically functional fragment (or fragment) of an antigen binding molecule is a type of antigen binding molecule containing a part (regardless of how that part is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in the full-length chain, but which are all still capable of specific binding to antigen. Such fragments are biologically active in that they bind to the target antigen and can be terminated by other antigen-binding molecules, including intact antibodies, to bind to the specified epitope. In some embodiments, the fragments are neutralizing fragments. In some embodiments, the fragments may block or reduce FLT3 activity. In one aspect, such a fragment will retain at least one CDR present in a full-length light or heavy chain, and in some embodiments, will contain a single heavy and/or light chain or portion thereof. These fragments can be obtained through recombinant DNA techniques, or can be obtained through enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.

Immunologically functional fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab') ₂ , etc.), one or more CDRs, diabodies (heavy chain variable domain on the same polypeptide as the light chain variable domain linked by a short peptide linker that is too short to allow association between two domains of the same chain), domain antibodies and single chain antibodies. These fragments may be obtained from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. As will be appreciated by one skilled in the art, the antigen binding molecule may include non-protein components.

Variants of antigen binding molecules are also included within the scope of the present invention, for example, a light chain variable region and/or a heavy chain variable region, each of which is at least 70-80%, 80-85%, 85-90%, 90- 95%, 95-97%, 97-99%, or greater than 99% identical to the amino acid sequences of the sequences described herein. In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant forms contain two identical light chains and two identical heavy chains (or subparts thereof). One skilled in the art will readily determine suitable variants of antigen binding molecules using well known techniques as described herein. In some embodiments, one skilled in the art will be able to identify suitable regions of the molecule that can be modified without destroying activity by targeting regions that are not considered important for activity.

In some embodiments, the polypeptide structure of the antigen-binding molecules is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, mini-antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, fusions antibodies (sometimes referred to herein as antibody conjugates), and fragments thereof, respectively. In some embodiments, the antigen-binding molecule contains or consists of avimers.

In some embodiments, the antigen binding molecule for FLT3 is administered separately. In other embodiments, the antigen binding molecule for FLT3 is administered as part of a CAR, TCR, or other immune cell. In such immune cells, the antigen binding molecule for FLT3 may be under the control of the same promoter region or a separate promoter. In some countries

- 14 044866 ants of implementation of the gene-coding protein means and/or antigen-binding molecule for FLT3 can be in separate vectors.

The present invention further provides pharmaceutical compositions containing an antigen binding molecule for FLT3 together with a pharmaceutically acceptable diluent, carrier, solvent, emulsifier, preservative and/or adjuvant. In some embodiments, the pharmaceutical compositions will include more than one different antigen binding molecule for FLT3. In some embodiments, the pharmaceutical compositions will include more than one FLT3 antigen binding molecule, wherein the FLT3 antigen binding molecule binds to more than one epitope. In some embodiments, different antigen binding molecules will not compete with each other for binding to FLT3.

In other embodiments, the pharmaceutical composition may be selected for parenteral delivery, for inhalation, or for delivery through the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the person skilled in the art. In some embodiments, buffers are used to maintain physiological pH or a slightly lower pH of the composition, typically within the pH range of about 5 to about 8. In some embodiments, if parenteral administration is contemplated, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable an aqueous solution containing the required antigen binding molecule for FLT3, with or without additional therapeutic agents, in a pharmaceutically acceptable medium. In some embodiments, the parenteral injection medium is sterile distilled water in which the antigen binding molecule for FLT3, with or without at least one additional therapeutic agent, is formulated as a sterile isotonic solution properly preserved. In some embodiments, preparation may involve formulation of the desired molecule with polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes that can provide a controlled or sustained release of the product, which can then be delivered by injection of a sustained release agent. In some embodiments, implantable drug delivery devices may be used to administer the desired molecule.

In some embodiments, the antigen binding molecule is used as a diagnostic or confirmation tool. An antigen binding molecule can be used to examine the amount of FLT3 present in a sample and/or subject. In some embodiments, the diagnostic antigen-binding molecule is not neutralizing. In some embodiments, the antigen binding molecules disclosed herein are used or provided in a test kit and/or method for detecting FLT3 in tissues or cells of a mammal for the purpose of monitoring/diagnosis of a disease or disorder associated with changes in FLT3 levels. The kit may include an antigen binding molecule that binds to FLT3, together with means for determining the binding of the antigen binding molecule to FLT3, if present, and optionally protein levels of FLT3.

The antigen binding molecule will be further understood in view of the definitions and descriptions below.

The Fc region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. The two heavy chain moieties are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

The Fab fragment contains one light chain, CH1 and variable regions of one heavy chain. One heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab' fragment contains one light chain and part of one heavy chain, which contains the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a molecule F(ab') ₂ . The F(ab') ₂ fragment contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. The F(ab') ₂ fragment is thus composed of two Fab' fragments which are held together by a disulfide bond between the two heavy chains.

The Fv region contains variable regions from both the heavy and light chains, but does not contain constant regions.

Single chain variable fragment (scFv, also called single chain antibody) refers to Fv molecules in which the heavy and light chain variable regions have been linked through a flexible linker to form a single polypeptide chain that forms the antigen binding region. See PCT Application WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference in their entirety.

A bivalent antigen-binding molecule contains two antigen-binding regions. In no

- 15 044866 in which cases the two binding regions have the same antigenic specificities. Bivalent antigen-binding molecules can be bispecific. A multispecific antigen-binding molecule is a molecule that targets more than one antigen or epitope. A bispecific, dual specificity, or bifunctional antigen binding molecule is a hybrid antigen binding molecule or antibody, respectively, with two different antigen binding regions. The two binding regions of a bispecific antigen binding molecule will bind to two different epitopes, which may be on the same or different protein targets.

An antigen binding molecule is reported to specifically bind its target antigen if the dissociation constant ( _Kd ) is ~ ^1x10'7 M. An antigen binding molecule specifically binds to an antigen having high affinity if the Kd is 1-5x10-9 M, and having extremely high affinity if the Kd is 1-5x10'1° M. In one embodiment, the antigen binding molecule has a Kd of ^10'9 M. In one embodiment, the dissociation rate is <1x10'5. In other embodiments, antigen binding molecules will bind to human FLT3 at a Kd of from about ^10'7 M to ^10'13 M, and in yet another embodiment, antigen binding molecules will bind at a Kd of 1.0-5 ^x10'10 .

An antigen-binding molecule is reported to be selective if it binds to one target more strongly than it binds to a second target.

The term antibody refers to an intact immunoglobulin of any isotype or fragment thereof that can compete with the intact antibody for specific binding to a target antigen, and includes, for example, chimeric, humanized, fully human and bispecific antibodies. The antibody is a type of antigen-binding molecule described herein. An intact antibody will typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, for example, antibodies naturally occurring in camelids, which may contain only heavy chains. Antibodies can be formed solely from one source, or can be chimeric, that is, different parts of the antibody can be formed from two different antibodies, as further described below. Antigen-binding molecules, antibodies, or binding fragments can be produced in hybridomas, through recombinant DNA techniques, or through enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term antibody includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, its derivatives, variants, fragments and muteins, examples of which are described below. Moreover, unless expressly excluded, antibodies include monoclonal antibodies, bispecific antibodies, mini-antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as antibody conjugates) and their fragments, respectively.

Variable regions typically exhibit the same general structure of relatively conserved framework (FR) regions connected by 3 hypervariable regions (ie, CDRs). The CDRs of the two strands of each pair are typically aligned through framework regions that can mediate binding to a specific epitope. From the N-terminus to the C-terminus, the variable regions of both the light chain and the heavy chain typically contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. Conventionally, the CDR regions in the heavy chain are generally referred to as HC CDR1, CDR2 and CDR3. The CDR regions in the light chain are commonly referred to as HC CDR1, CDR2 and CDR3. The amino acid assignment for each domain usually follows the definitions of Kabat (Seqs of Proteins of Immunological Interest (NIH, Bethesda, MD (1987 and 1991)), or Chothia (J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).) Various methods can be used to identify or approximate CDR regions, including not only Kabat or Chothia, but also AbM determination.

The term light chain includes the full-length light chain and fragments thereof with sufficient variable region sequence to provide binding specificity. The full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The light chain variable region domain represents the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

The term heavy chain includes the full-length heavy chain and fragments thereof with sufficient variable region sequence to provide binding specificity. The full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The V _H domain is located at the amino terminus of the polypeptide, and the _CH domains are located at the carboxyl terminus, with CH3 closest to the carboxyl terminus of the polypeptide. The heavy chains can be any isotype, including IgG (including the IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including the IgA1 and IgA2 subtypes), IgM and IgE.

The term variable region or variable domain refers to the mild and/or severe portion

- 16,044,866 antibody chains, typically including approximately amino terminal 120 to 130 amino acids in the heavy chain and approximately amino terminal 100 to 110 amino acids in the light chain. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

Variability is not distributed evenly across all antibody variable domains; it is concentrated in subdomains of each of the variable regions of the heavy and light chains. Such subdomains are called hypervariable regions or complementarity determining regions (CDRs). The more conserved (ie, non-hypervariable) portions of the variable domains are called framework regions (FRMs or FRs) and provide a framework for the formation of an antigen-binding surface of six CDRs in three-dimensional space. The variable domains of naturally occurring heavy and light chains contain four FRM regions (FR1, FR2, FR3, and FR4), primarily using a β-sheet configuration connected by three hypervariable regions that form loops connecting the β-sheet structure, and in some cases forming part of its structure. The hypervariable regions on each chain are held together in close proximity to the FRM and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen binding site (see Kabat et al., loc. cit.).

The term CDR, in the singular and plural, refers to the complementarity determining region. Three such regions impart binding character to the light chain variable region (CDR-L1, CDR-L2, and CDR-L3), and another three impart binding character to the variable heavy chain region (CDRH1, CDR-H2, and CDR-H3). CDRs contain most of the residues responsible for the specific interactions of the antibody with the antigen and, therefore, contribute to the functional activity of the antibody molecule: they are the main determinants of antigen specificity.

Specific definitions of CDR boundaries and lengths are tied to various classification and numbering systems. Therefore, CDRs may be determined by Kabat, Chothia, contact definitions, or any other boundary definitions, including the numbering system described herein. Despite their different boundaries, each of these systems has some degree of overlap in what constitutes the so-called hypervariable regions in variable sequences. Thus, CDR definitions under these systems may differ in the length and boundaries of the regions relative to the adjacent frame region. See, for example, Kabat (an approach based on interspecies sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes) and/or MacCallum (Kabat et al., loc. cit.; Chothia et al., J Mol Biol 1987 196:901-917 and MacCallum et al J Mol Biol 1996 262:732). Another standard for antigen binding site characterization is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. B: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). To the extent that the two residue identification methods identify regions of overlapping but not identical regions, they can be combined to define a hybrid CDR. However, numbering according to the so-called Kabat system is preferred.

Typically, CDRs form a loop structure, which can be classified as a canonical structure. The term canonical structure refers to the backbone conformation that is determined by the antigen binding loops (CDRs). Comparative structural studies revealed that five of the six antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the twist angles of the polypeptide backbone. Thus, corresponding loops between antibodies may have fairly similar three-dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature , 1989, 342: 877; Martin and Thornton, J. Mol. Biol., 1996, 263: 800). In addition, there is a relationship between the adopted loop structure and the surrounding amino acid sequences. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop, as well as within the conserved framework (i.e., outside the loop). Thus, assignment of a particular canonical class can be based on the presence of these key amino acid residues.

The term canonical structure may also include considerations regarding the linear sequence of the antibody, for example, as cataloged by Kabat (Kabat et al., loc. cit.). The Kabat numbering scheme is a widely accepted standard for numbering amino acid residues of an antibody variable domain in a consistent manner and is the preferred scheme used in the present invention, as also mentioned elsewhere herein. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, similar differences not fully captured by Kabat's numbering can be described by the numbering system of Chothia et al. and/or disclosed by other techniques, such as crystallography and two- or three-dimensional computer modeling.

- 17 044866

Accordingly, a given antibody sequence can be assigned to a canonical class, which allows, among other things, the identification of relevant framework sequences (eg, based on the desire to include many canonical structures in the library). Kabat amino acid sequence numbering and structural considerations as described in Chothia et al., loc. cit., as well as their significance for the interpretation of canonical aspects of antibody structure, are described in the relevant literature. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known in the art. For an overview of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988

Light chain CDR3 and, in particular, heavy chain CDR3 may represent the most important determinants of antigen binding in the light and heavy chain variable regions. In some antibody designs, the heavy chain CDR3 appears to be the major contact region between the antigen and the antibody. In vitro selection schemes in which only CDR3 is changed can be used to alter the binding properties of an antibody or determine which residues contribute to antigen binding. Therefore, CDR3 tends to be the greatest source of molecular diversity at the antibody binding site. H3, for example, can be as short as two amino acid residues or as long as 26 amino acids.

The term neutralizing refers to an antigen-binding molecule, scFv or antibody, respectively, that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be accomplished, for example, by directly blocking the binding region on the ligand, or by binding to the ligand and altering the binding ability of the ligand through indirect means (such as structural or energetic changes in the ligand). In some embodiments, the term may also refer to an antigen-binding molecule that prevents the protein to which it binds from performing a biological function.

The term target or antigen refers to a molecule or part of a molecule that is capable of being bound by an antigen-binding molecule. In some embodiments, the target may have one or more epitopes.

The term compete, when used in the context of antigen-binding molecules that compete for the same epitope, means competition between antigen-binding molecules, as determined by an assay in which the antigen-binding molecule being tested (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g. , reduces) the specific binding of said antigen-binding molecule to the antigen. Various types of competitive binding assays can be used to determine whether an antigen-binding molecule competes with another molecule, such as direct or indirect solid-phase radioimmunoassay (RIA), direct or indirect solid-phase enzyme immunoassay (EIA), multilayer competition assay (Stahli et al ., 1983, Methods in Enzymology 9:242-253); direct solid-phase biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619), direct solid-phase labeled assay, direct solid-phase multilayer assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeled solid phase RIA using 1-125 labels (Morel et al., 1988, Molec. Immunol. 25:7-15); direct biotin-avidin solid phase EIA (Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). The term epitope includes any determinant capable of being bound by an antigen-binding molecule, for example, a scFv, antibody or immune cell of the present invention. An epitope is a region of an antigen that is bound by an antigen-binding molecule targeting that antigen, and an antigen is a protein that includes specific amino acids that directly contact the antigen-binding molecule.

As used herein, the terms labeled or labeled refer to the introduction of a detectable marker, for example, by introducing a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker, or an enzymatic activity that can be detected by optical or colorimetric methods). In some embodiments, the label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used.

In accordance with the present invention, the on-off type or other types of control switching techniques may be included herein. These techniques may involve the use of dimerization domains and optional activators of such dimerization domains. These techniques include, for example, those described by Wu et al., Science 2014 350 (6258) using FKBP/Rapalog dimerization systems in certain cells, the contents of which are incorporated herein by reference in their entirety. Additional dimerization technology is described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336, and also in patents

- 18 044866

US No. 5830462; 5834266; 5869337 and 6165787, the contents of which are also incorporated herein by reference in their entirety. Additional dimerization pairs may include cyclosporin-A/cyclophilin receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoid/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. The following examples of dimerization technology can be found, for example, in WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, US patent No. 8486693, US 2014/0171649 and US 2012/0130076, the contents of which are also incorporated herein by reference in its entirety.

Treatment methods

Using adoptive immunotherapy, the original T cells can be (i) removed from the patient, (ii) genetically engineered to express a chimeric antigen receptor (CAR) that binds to at least one tumor antigen, or (iii) expanded ex vivo in a large population of engineered T cells, and (iv) reintroduced to the patient. See, for example, US Pat. Nos. 7,741,465 and 6,319,494 to Eshhar et al. (Cancer Immunol, supra); Krause et al. (supra); Finney et al. (supra). Once the engineered T cells are reintroduced into a patient, they mediate an immune response against cells expressing the tumor antigen. See, for example, Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626). This immune response includes secretion of IL-2 and other cytokines by T cells, clonal expansion of T cells recognizing tumor antigen, and T cell-mediated specific killing of target-positive cells. See Hombach et al., Journal of Immun. 167: 6123–6131 (2001).

Therefore, in some aspects, the present invention includes a method of treating or preventing a condition associated with undesirable and/or elevated levels of FLT3 in a patient, comprising administering to a patient in need thereof an effective amount of at least one isolated antigen binding molecule, CAR or TCR, disclosed in this document.

Methods for treating diseases or disorders, including cancer, are provided. In some embodiments, the present invention relates to generating a T cell-mediated immune response in a subject, comprising administering to the subject an effective amount of engineered immune cells of the present application. In some embodiments, the T Cell-mediated immune response is directed against target cells or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some embodiments, the target cell is a tumor cell. In some aspects, the present invention contains a method of treating or preventing the formation of a malignant tumor, the method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen binding molecule described herein. In some aspects, the present invention comprises a method of treating or preventing the formation of a malignant tumor, the method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell contains at least one chimeric antigen receptor, T cell receptor and/or an isolated antigen binding molecule as described herein.

In some aspects, the present invention includes a pharmaceutical composition comprising at least one antigen binding molecule as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

Antigen-binding molecules, CARs, TCRs, immune cells, etc. The present invention may be used to treat myeloid diseases, including, but not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, acute promyelocytic leukemia (APL) ), acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma, or combinations thereof. Additional diseases include inflammatory and/or autoimmune diseases, such as rheumatoid arthritis, psoriasis, allergies, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and celiac disease.

It should be understood that target doses for CAR ⁺ /CAR-T ⁺ /TCR ⁺ cells may vary in the range of 1 x 10 ⁶ -2 x ^{10 10} cells/kg, preferably 2 x 10 ⁶ cells/kg, more preferably. It should be understood that dosages above and below this range may be suitable for certain subjects, and suitable dosage levels, if necessary, can be determined by the attending physician. In addition, multiple doses of cells in accordance with the present invention may be provided.

Also provided are methods for reducing the size of a tumor in a subject, comprising administering to the subject a formulated cell of the present invention, wherein the cell comprises a chimeric antigen receptor, a T-cell receptor, or a chimeric antigen receptor-based T-cell receptor comprising an antigen-binding molecule that binds to a tumor antigen. In some

- 19 044866 embodiments, the subject has a solid tumor or a blood malignancy, such as lymphoma or leukemia. In some embodiments, the engineered cell is delivered to the tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject.

In some embodiments, the engineered cells are autologous T cells. In some embodiments, the designed cells are allogeneic T cells. In some embodiments, the engineered cells are heterologous T cells. In some embodiments, the developed cells of this application are transfected or transformed in vivo. In some embodiments, the developed cells of this application are transfected or transformed ex vivo.

The methods may further include administering one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is a lymphocyte function-reducing (pre-adapting) chemotherapeutic agent. Advantageous pretreatment regimens, together with corresponding advantageous biomarkers, are described in provisional patent applications 62/262143 and 62/167750, which are incorporated herein by reference in their entirety. They describe, for example, methods for adapting a patient in need of T-cell therapy, including administering to the patient certain preferential doses of cyclophosphamide (from 200 mg/m ² /day to 2000 mg/m ² /day) and certain doses of fludarabine (from 20 mg/ ^m2 /day up to 900 mg/ ^m2 /day). A preferred dosage regimen involves treating the patient by administering approximately 500 mg/m ² /day to the patient daily. cyclophosphamide and approximately 60 mg/m ² /day. fludarabine for three days before administering a therapeutically effective amount of engineered T cells to the patient.

In other embodiments, each of an antigen binding molecule, transformed (or otherwise engineered) cells (eg, CAR or TCR), and a chemotherapeutic agent is administered in an amount effective to treat a disease or condition in a subject.

In some embodiments, compositions containing CAR-expressing immune effector cells disclosed herein can be administered together with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan and piposulfan; azaridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards, such as chlorambucil, chlornaphasine, chlorphosphamide, estramucin, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, activemycin, outramycin, azaserin, bleomycins, cactinomycin, calicheymycin, carabicin, carminomycin, carcinophilin, chromomycins, dactinomycins, daunorubicin, detorubicin, 6-diazo-5oxo-b-norleucine, doxorubicin, epirubicin, esorubicin, idarubi qing, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycins, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, cinostatin, zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancesitabine, azacitidine, 6azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquon; elformitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentosatin; phenomet; pirarubicin; podophyllic acid; 2-ethylhydrazine; procarbazine; PSK®; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and dexetaxel (TAXOTERE®, RhonePoulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novatron; teniposide; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™, (aliretinoin); ONTAK™ (denileukin diftitox); esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones related to

- 20 044866 especially tumors, for example, antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. If necessary, combinations of chemotherapeutic agents are also administered, including without limitation CHOP, i.e. cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorbicine), vincristine (Oncovin®), and prednisone.

In some embodiments, the chemotherapeutic agent is administered concurrently with or within a week of administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapy agent is administered for 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 weeks to 12 months after administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some embodiments, the methods further include administering two or more chemotherapeutic agents.

A large number of additional therapeutic agents can be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab and atezolizumab.

Additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emastatin (KADCYLA ®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitin ib, masitinib , pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintendanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib b, dasatinib, nilotinib, ponatinib, radotinib, bosutinib , lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, Hedgehog signaling pathway inhibitors such as Sonide gib and vismodegib, inhibitors CDKs, such as a CDK inhibitor (palbociclib).

In additional embodiments, a composition comprising a CAR immune cell may be administered with an anti-inflammatory agent. Anti-inflammatory drugs or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary pain relievers include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (eg, CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (eg, etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE ®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies, and recombinant forms of the molecules. Illustrative DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofine) and intramuscular administration) and minocycline.

In some embodiments, the compositions described herein are administered together with a cytokine. Cytokine, as used herein, is intended to refer to proteins released by one population of cells that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines and common polypeptide hormones. In addition to cytokines, growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone are included; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs), such as NGF-beta;

- 21 044866 platelet growth factor; transforming growth factors (TGFs), such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta and -gamma; colony-stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12; IL-15, tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the original sequence cytokines.

In some aspects, the present invention includes an antigen binding molecule that binds FLT3 at a Kd of less than 100 pM. In some embodiments, the antigen binding molecule binds at a Kd of less than 10 pM. In other embodiments, the antigen binding molecule binds at a Kd of less than 5 pM.

Methods of obtaining

A variety of techniques, polypeptides, vectors, antigen binding molecules, immune cells, compositions, and the like can be used to produce polynucleotides. according to the present invention.

Before manipulating or genetically modifying in vitro immune cells described herein, the cells may be obtained from a subject. In some embodiments, the immune cells include T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, site of infection tissue, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using a variety of techniques known to one of ordinary skill in the art, for example, the FICOLL™ department. The cells may preferably be obtained from the circulating blood of an individual via apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for further processing. Cells can be washed with PBS. As will be appreciated, a washing step may be employed, for example, using a semi-automated flow centrifuge, such as a Cobe™ 2991 Cell Processor, Baxter CytoMate™ or the like. After washing, the cells can be resuspended in various biocompatible buffers or other saline solution with or without buffer. In some embodiments, unwanted components of the apheresis sample may be removed.

In some embodiments, T cells are isolated from PBMCs by disrupting red blood cells and reducing monocyte function, for example, using PERCOLL™ gradient centrifugation. A specific subset of T cells, for example, CD28+, CD4 ⁺ , CD8 ⁺ , CD45RA+, and CD45RO ⁺ T cells can be further isolated through positive or negative selection techniques known in the art. For example, enrichment of a T cell population through negative selection can be achieved with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells through negative selection, the monoclonal antibody mixture typically includes antibodies to CD14, CD20, CD1lb, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to isolate populations of cells of interest for use in the present invention.

PBMCs can be used directly for genetic modification with immune cells (eg, CAR or TCR) using the methods described herein. In some embodiments, after isolation of PBMCs, T lymphocytes may be further isolated and both cytotoxic and helper T lymphocytes may be sorted into primary, memory, and effector T cell subsets, either before or after genetic modification and/or expansion .

In some embodiments, CD8+ cells are further sorted into primary, central storage, and effector cells by identifying cell surface antigens that are associated with each of these CD8+ cell types. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, the central memory T cells are CD45RO+, CD62L+, CD8 ⁺ -T -cells. In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and

- 22 044866

CD127, and positive for granzyme B and perforin. In some embodiments, the CD4 ⁺ T cells are further sorted into subpopulations. For example, helper CD4 ⁺ T cells can be sorted into primary, central storage and effector cells by identifying cell populations that have cell surface antigens.

Immune cells, such as T cells, can be genetically modified after isolation using known methods, or immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro before genetic modification. In another embodiment, immune cells, such as T cells, are genetically modified with chimeric antigen receptors described herein (e.g., transformed with a viral vector containing one or more nucleotide sequences encoding a CAR) and then activated and/or expanded in vitro . Methods for activating and expanding T cells are known in the art and are described, for example, in US Pat. No. 6,905,874; US Patent No. 6867041; US Patent No. 6797514; and PCT WO 2012/079000, the contents of which are incorporated herein by reference in their entirety. Typically, such methods involve contacting PBMCs or isolated T cells with a simulator and a co-stimulator, such as antibodies to CD3 and CD28, typically attached to a bead or other surface, in a culture medium with appropriate cytokines such as IL -2. Antibodies to CD3 and CD28 attached to the same granule act as a surrogate antigen presenting cell (APC). One example is The Dynabeads® system, a CD3/CD28 stimulator/activator system for physiological activation of human T cells.

In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US Pat. No. 6,040,177; US Patent No. 5827642; and WO 2012129514, the contents of which are incorporated herein by reference in their entirety.

Certain methods for producing the constructs and engineered immune cells of the present invention are described in PCT application PCT/US15/14520, the contents of which are incorporated herein by reference in their entirety. Additional methods for producing constructs and cells can be found in US Provisional Patent Application No. 62/244,036, the contents of which are incorporated herein by reference in their entirety.

It should be understood that PBMC may additionally include other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of a chimeric receptor disclosed herein can be introduced into a population of T cells, NK cells or NKT cells of a human donor. Successfully converted T cells that carry the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells and then further expanded to increase the number of these CAR-expressing T cells, in addition to activating the cells using anti-CD3 and anti-IL-cells. 2 or other methods known in the art, as described elsewhere herein. Standard procedures may be used to cryopreserve CAR-expressing T cells for storage and/or retrieval for use in a human subject. In one embodiment, in vitro transformation, culture and/or expansion of T cells is carried out in the absence of non-human animal derived products such as fetal bovine serum and fetal bovine serum.

To clone polynucleotides, a vector can be introduced into a host cell (an isolated host cell) to cause the vector itself to replicate and thereby amplify copies of the polynucleotide contained therein. Cloning vectors may contain sequence components, which typically include, but are not limited to, an origin of replication, promoter sequences, transcription initiation promoters, enhancer sequences, and selectable markers. These elements can be selected accordingly by one skilled in the art. For example, the origin of replication may be selected to facilitate autonomous replication of the vector in the host cell.

In some embodiments, the present disclosure provides isolated host cells containing a vector provided herein. Host cells containing the vector may be useful in expressing or cloning the polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. Suitable pokaryotic cells for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive bacteria organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

The vector can be introduced into a host cell using any suitable methods known in the art, including, without limitation, DEAE-dextran-mediated delivery, deposition method

- 23 044866 calcium phosphate, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microparticle bombardment, receptor mediated gene delivery, polylysine, histone, chitosan and peptide mediated delivery. Standard methods for transfecting and transforming cells to express a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors for genetically modifying a donor population of immune effector cells, wherein each vector encodes a different CAR disclosed herein. The resulting converted immune effector cells form a mixed population of engineered cells, with a proportion of engineered cells expressing more than one different CAR.

In one embodiment, the present invention provides a method for storing genetically engineered cells expressing a CAR or TCR that target the FLT3 protein. It involves cryopreservation of immune cells so that the cells remain viable after thawing. A proportion of immune cells expressing CARs can be cryopreserved by methods known in the art to provide a continuous source of such cells for future treatment of patients suffering from cancer. If necessary, cryopreserved transformed immune cells can be thawed, grown and expanded into more such cells.

As used herein, the term cryopreservation refers to the preservation of cells by cooling to temperatures below freezing, for example (typically) 77 K or -196°C (the boiling point of liquid nitrogen). At temperatures below zero, cryoprotectants are often used to prevent damage to preserved cells due to freezing at low temperatures or heating to room temperature. Cryopreservation agents and optimal cooling rates can protect cells from damage. Cryoprotectants that can be used in accordance with the present invention include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock & Bishop, Nature (1959); 183: 1394-1395; Ashwood-Smith, Nature (1961); 190: 1204-1205 ), glycerol, polyvinylpyrrolidine (Rmfret, Ann. N.Y. Acad. Sci. (1960); 85: 576) and polyethylene glycol (Sloviter & Ravdin, Nature (1962); 196: 48). The preferred cooling rate is 1-3°C/min.

The term essentially pure is used to indicate that the given component is present at a high level. The component is advantageously the main component present in the composition. Preferably it is present at a level of greater than 30%, greater than 50%, greater than 75%, greater than 90%, or even greater than 95%, which level is determined on a dry weight/dry weight basis relative to the total weight of the composition in question. At very high levels (eg, levels greater than 90%, greater than 95%, or greater than 99%), the component may be considered to be in its pure form. The biologically active substances of the present invention (including polypeptides, nucleic acid molecules, antigen binding molecules, fragments) may be presented in a form that is substantially free of one or more contaminants to which the substance may otherwise be associated. If the composition is substantially free of a given contaminant, then the contaminant will be at a low level (eg, less than 10%, less than 5%, or less than 1% on a dry weight/dry weight basis, as stated above).

In some embodiments, the cells are formulated by first collecting them from their culture medium and then washing and concentrating the cells in a medium and storage system suitable for administration (a pharmaceutically acceptable carrier) in a treatment-effective amount. Suitable infusion media can be any isotonic solution of the media, typically saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's may also be used. The infusion medium may be supplemented with human serum albumin.

The number of cells in the composition required for treatment is typically at least 2 cells (e.g., at least 1 CD8+ central storage T cell and at least 1 CD4+ helper subset T cell) or more typically greater than 10 ² cells, and not more than 106, not more than 10 ⁸ or 10 ⁹ cells inclusive and may be more than 10 ¹⁰ cells. The number of cells will depend on the desired application for which the composition is intended and the type of cells included herein. The required cell density is typically greater than 106 cells/ml and typically greater than 10 ⁷ cells/ml, typically 10 ⁸ cells/ml or more. A clinically appropriate number of immune cells can be divided into multiple infusions that total equal to or greater than 105, 106, ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , ¹⁰¹⁰ , ¹⁰¹¹ or ¹⁰¹² cells. In some aspects of the present invention, particularly since all administered cells will be redirected to a specific target antigen (FLT3), lower numbers of cells may be administered, in the range of 10 ⁶ /kg (10 6-10 ¹¹ per patient). The CAR therapeutics may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient being treated.

The CAR-expressing cell populations of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components.

- 24 044866 components, for example IL-2 or other cytokines, or cell populations. The pharmaceutical compositions of the present invention may contain a CAR or TCR expressing cell population, for example, T cells, as described herein, in combination with one or more pharmaceutically or acceptable carriers, diluents or excipients. Such compositions may contain buffers, for example, neutral buffer solution, phosphate buffer solution, etc.; hydrocarbons such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; auxiliary agents (for example, aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

Pharmaceutical compositions (solutions, suspensions or the like) may include one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride solution, fixed oils such as synthetic mono- or diglycerides, which can act as solvents or suspending media, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusters such as sodium chloride or dextrose. The drug for parenteral use may be contained in ampoules, disposable syringes or multi-dose containers made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

It should be understood that adverse events can be minimized by transforming immune cells (containing one or more CARs or TCRs) with a suicide gene. It may also be necessary to introduce an inducible switch or accelerator into the immune cells. Suitable techniques include the use of inducible caspase-9 (US patent application 2011/0286980) or thymidine kinase, before, after or simultaneously with the transformation of cells using the CAR construct of the present invention. Additional methods for introducing suicide genes and/or switches include TALENS, zinc finger domains, RNAi, siRNA, shRNA, antisense technology and other techniques known in the art.

It should be understood that the descriptions herein are illustrative and explanatory only and do not limit the present invention as claimed. In this application, the use of singular forms includes the plural unless expressly stated otherwise.

The section titles used herein are for organizational purposes only and are not intended to limit the subject matter described. All documents or portions of documents cited in this application, including, without limitation, patents, patent applications, articles, books and scientific papers are incorporated herein by reference in their entirety for any purpose. As used in this disclosure, the following terms, unless otherwise specified, are to be understood as having the following meaning:

In this application, the use of either means and/or, unless otherwise indicated.

In addition, the use of the term including, as well as other forms such as includes and inclusive, is non-limiting. In addition, terms such as element or component cover both elements and components containing a single unit and elements and components that contain more than one subunit, unless specifically stated otherwise.

The term FLT3 activity includes any biological effect of FLT3. In some embodiments, FLT3 activity includes the ability of FLT3 to interact or bind to a substrate or receptor.

The term polynucleotide, nucleotide or nucleic acid includes both single-stranded and double-stranded nucleotide polymers. The nucleotides constituting a polynucleotide may be ribonucleotides or deoxyribonucleotides, or a modified form of any type of nucleotide. These modifications include base modifications such as bromuridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide modifications such as phosphothioate, phosphodithioate, phosphoselenoate, phosphodiselenoate, phosphoanilotioate, phosphoaniladate and phosphoamidate.

The term oligonucleotide refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single-stranded or double-stranded, for example for use in constructing a mutant gene. The oligonucleotides may be sense or antisense oligonucleotides. The oligonucleotide may include a label, including a radioisotope label, a fluorescent label, a hapten or an antigenic label, for a detection assay. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.

The term reference sequence refers to a polynucleotide sequence that

- 25 044866 which affects the expression and processing of the coding sequence to which it binds via ligand. The nature of such a control sequence may depend on the host organism. In specific embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding region, and a transcription termination sequence. For example, control sequences for eukaryotes may include promoters including one or more recognition regions for transcription factors, transcription enhancer sequences, and transcription termination sequences. Control sequences may include leader sequences (signal peptides) and/or cell fusion sequences.

As used herein, the term operably coupled means that the components to which the term applies are in a relationship that allows them to perform their inherent functions under suitable conditions.

The term vector means any molecule or particle (eg, nucleic acid, plasmid, bacteriophage or virus) used to transfer protein-coding information into a host cell. The term expression vector or expression construct refers to a vector that is suitable for transforming a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation and, if introns are present, effects on RNA splicing of the coding region operably linked thereto.

The term host cell refers to a cell that has been transformed, or capable of transformation, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the original cell, whether the progeny is identical in terms of morphology or genetic design to the original cell or not, as long as the gene of interest is present.

The term transformation refers to a change in the genetic characteristics of a cell, and a cell has been transformed if it has been modified to contain new DNA or RNA. For example, a cell is transformed if it is genetically modified from its original state by introducing new genetic material through transfection, transformation, or other techniques. Following transfection or transformation, the transforming DNA may recombine with the cell's DNA by physical integration into the cell's chromosome, or may be maintained transiently as an episomal element without replication, or may undergo independent replication as a plasmid. A cell is considered stably transformed if the transforming DNA undergoes replication with cell division.

The term transfection refers to the uptake of foreign or exogenous DNA by a cell. A number of transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

The term transduction refers to the process in which foreign DNA is introduced into a cell via a viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

The terms polypeptide or protein refer to a macromolecule with the amino acid sequence of a protein, including deletions, additions and/or substitutions of one or more amino acids of the original sequence. The terms polypeptide and protein specifically encompass FLT3 antigen binding molecules, antibodies or sequences having deletions, additions and/or substitutions of one or more amino acids of the antigen binding protein. The term polypeptide fragment refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion from the full-length parent protein. Such fragments may also contain modified amino acids compared to the original protein. Suitable polypeptide fragments include immunologically functional fragments of antigen binding molecules. Suitable fragments include, but are not limited to, one or more CDR regions, heavy and/or light chain variable domains, portions of other portions of the antibody chain, and the like.

The term isolated means (i) free from at least a few other proteins with which it might normally occur, (ii) essentially free from other proteins from the same source, such as from other species, (iii) separated of at least about 50% of the polynucleotides, lipids, hydrocarbons or other materials to which it is naturally associated, (iv) operably linked (by covalent or non-covalent interaction) to a polypeptide to which it is not naturally associated, or ( v) does not occur naturally.

A variant polypeptide (eg, antigen binding molecule or antibody) contains an amino acid sequence in which one or more amino acid residues are introduced

- 26 044866 inside, removed from and/or substituted in the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

The term identity refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by sequence alignment and comparison. Percent identity refers to the percentage of identical residues between amino acids or nucleotides in the molecules being compared and is calculated based on the size of the smallest molecule being compared. For these calculations, alignment gaps (if any) are preferably adjusted through a specific mathematical model or computer program (ie, algorithm).

To calculate percent identity, the compared sequences are usually aligned using the method that provides the greatest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG software package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis. ). The GAP computer algorithm is used to align two polypeptides or polynucleotides for which the percentage of sequence identity must be determined. Sequences are aligned to optimally match their corresponding amino acid or nucleotide (matched population determined by the algorithm). In some embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci U.S.A. 89:10915-10919 for the comparison matrix BLOSUM 62) is also used in the algorithm.

As used herein, the 20 common (eg, naturally occurring) amino acids and their abbreviations follow common usage. See Immunology - A Synthesis (2nd Edition, Golub and Gren, Eds., Sinauer Assoc, Sunderland, Mass. (1991)), incorporated herein by reference for all purposes. Stereoisomers (eg, D-amino acids) of the 20 common amino acids, unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid and other atypical amino acids may also be useful components for the polypeptides of the present invention. Examples of atypical amino acids include the following: 4-hydroxyproline, γ-carboxyglutamate, epsilonΝ,Ν,Ν-trimethyllysine, e-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N- methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide designation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and conventions.

Conservative amino acid substitutions may involve non-naturally occurring amino acid residues that are typically introduced by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid fragments. Naturally occurring residues can be divided into classes based on general side chain properties:

a) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c) acidic: Asp, Glu;

d) basic: His, Lys, Arg;

e) residues that affect chain orientation: Gly, Pro and

f) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve replacing a member of one of these classes with a member of another class. Such substituted residues may be introduced, for example, into regions of a human antibody that are homologous to non-human antibodies or into non-homologous regions of the molecule.

In effecting changes to the antigen binding molecule costimulatory or activating domains of the designed T cell, in accordance with some embodiments, the hydrophobicity index may be considered. Each amino acid was assigned a hydrophobicity index based on its hydrophobicity and alteration characteristics. They are the following: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). See Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that some amino acids can be replaced by other amino acids with a similar hydrophobicity index or score and still retain similar biological activity. It will also be appreciated by those skilled in the art that substitution of such amino acids can be effectively made on the basis of hydrophilicity, particularly if the biologically functional protein or peptide so created is intended

- 27 044866 for use in immunological embodiments, as in this case. Exemplary amino acid substitutions are presented in table. 2.

table 2

Illustrative Preferred

Original substitution substitution balances

Ala Vai, Leu, He Vai Arg Lys, Gin, Asn Lys Asn Gin Gin Asp Glu Glu Cys Ser, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gin, Lys, Arg Arg He Leu, Vai, Met, Ala, Phe, norleucine Leu Leu Hop Leucine, He, Vai, Met, Ala, Phe He Lys Arg, 1,4-diaminobutyric Arg Acid, Gin, Asn Met Leu, Phe, He Leu Phe Leu, Vai, lie, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Vai lie, Met, Leu, Phe, Leu Ala, norleucine

The term derivative refers to a molecule that involves a chemical modification other than the insertion, deletion or substitution of amino acids (or nucleic acids). In some embodiments, the derivatives contain covalent modifications, including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic moieties. In some embodiments, a chemically modified antigen binding molecule may have a longer half-life in the bloodstream than an antigen binding molecule that is not chemically modified. In some embodiments, the derived antigen-binding molecule is covalently modified to include one or more water-soluble polymeric attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

Peptide analogues are widely used in the pharmaceutical industry as non-peptide drugs with properties similar to matrix peptide drugs. These types of non-peptide compounds are called peptide mimetics or peptidomimetics. Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber & Freidinger, TINS, p. 392 (1985) and Evans et al., J. Med. Chem., 30:1229 (1987), which is incorporated herein by reference for all purposes.

The term therapeutically effective amount refers to the amount of antigen-binding molecule to FLT3 that is determined to provide a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertainable by one of ordinary skill in the art.

The terms patient and subject are used interchangeably and include human subjects and non-human animal subjects, as well as subjects with previously diagnosed disorders, without previously diagnosed disorders, receiving medical care, at risk of developing disorders, etc.

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The term treat and treatment includes therapeutic treatments, prophylactic treatments, and uses that reduce a subject's risk of developing a disorder or other risk factor. Treatment does not require complete cure of the disorder and covers embodiments in which symptoms or underlying risk factors are reduced. The term prevent does not require 100% elimination of the possibility of a phenomenon. Rather, it means that the likelihood of the phenomenon occurring has been reduced in the presence of the compound or method.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reaction and purification procedures may be carried out in accordance with the manufacturer's instructions or as commonly used in the art, or as described herein. The above techniques and procedures can generally be carried out in accordance with conventional methods well known in the art, and as described in the various general and more specific reference materials that are cited and discussed herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for all purposes.

The following sequences will further illustrate the present invention.

CD28T DNA extracellular, transmembrane, intracellular

CTTGATAATGAAAAGTC

AAACGGAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCC

TGGTCCATCCAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTA CTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAGAAGCCG CCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAA ACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEQ Ш NO: P

CD28T extracellular, transmembrane, intracellular AA:

LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV

AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRS (SEQIDNO:2)

CD28T DNA - extracellular

CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAA

GCACCTCTGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA (SEQ Ш NO: 3)

CD28T AA - extracellular

LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP (SEQ Ш NO: 4)

CD28 DNA transmembrane domain

TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCG

TGGCTTTTATAATCTTCTGGGTT (SEQ Ш NO: 5)

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CD28 AL transmembrane domain:

FWVLVVVGGV LACYSLLVTV AFIIFWV (SEQ ID NO: 6)

CD28 DNA intracellular domain:

AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCG CCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGC CTATCGGAGC (SEQ ID NO: 7)

CD28 AA intracellular domain

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 8)

CD3 zeta DNA

AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCA ACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGC GCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGA GGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAG GCATGAAAGGAGAG CGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACT CAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAG G (SEO ID NO: 9)

CD3 zeta AA

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ Ш NO: 10)

CD28 DNA

ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGTACCAT CATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCCTCTTCCCCGGGCCATCAAA GCCC (SEQ ID NO: 11)

CD28AA

IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKP (SEQIDNO: 12)

CD8 DNA extracellular and transmembrane domain

GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGTTCTTGC CGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCTCCTACCATCG CTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCAGGGGGCGCTG TTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACCCCTGGCCG

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GAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGTAATCACAGGA AC (SEQ Ш NO: 13)

CD8 AA extracellular and transmembrane domain

AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 14)

Clone 10E3 NS DNA

CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTGGTGAAACCCACAGAGACCCTCAC GCTGACCTGCACCGTCTCTGGGTTCTCACTCATCAATGCTAGAATGGGTGTGAGCTG GATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGC CGAAAAATCGTACAGGACATCTCTGAAGAGCAGGCTCACCATCTCCAAGGACACCT CCAAAAGCCA GGTGGTCCTTACCATGACCAACATGGACCCTGTGGACACAGCCACA TATTACTGTGCACGGATACCAGGCTACGGTGGTAACGGGGACTACCACTACTACGGT ATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 15)

Clone 10E3 NS AA - CDRs underlined

QVTLKESGPVLVKPTETLTLTCTVSGFSLINARMGVSWIRQPPGKALEWLAHIFSNAEKS YRTSLKSRLTISKDTSKSOVVLTMTNMDPVDTATYYCARIPGYGGNGDYHYYGMDVW GQGTTVTVSS (SEQ ID NO: 16)

Clone 10E3 NS AACDR1:

NARMGVS (SEQ Ш NO: 17)

Clone 10E3 NS AA CDR2: HIFSNAEKSYRTSLKS (SEP ID NO: 18)

Clone 10E3 NS AA CDR3: IPGYGGNGDYHYYGMDV (SEP ID NO: 19)

Clone 10E3 LC DNA

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTCTAGGAGACAGAGTC ACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCA GAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTTCATCCACTTTGCAAAGTGG GGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGGTTCACTCTCACAATCA GCAGCCTGCAGC CTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAATTTCC CGTGGACGTTCGGTCAGGGAACGAAGGTGGAAATCAAACGA (SEQ ID NO: 20)

Clone 10E3 LC AA (CDR underlined)

DIOMTOSPSSLSASLGDRVTITCRASQGIRNDLGWYOOKPGKAPKRLIYASSTLOSGVPS

RFSGSGSGTEFTLTISSLOPEDFATYYCLQHNNFPWTFGOGTKVEIKR (SEQ ID NO: 21)

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Clone 10EZ LC CDR1 AL:

RASQGIRNDLG (SEP ID NO: 22)

Clone 10E3 LC CDR2 AA: ASSTLQS (SEP ID NO: 23)

Clone 10E3 LC CDR3 AA: LpHNNFPWT (SEP ID NO: 24)

Clone 2E7 NS DNA

CAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTGGTGAAACCCACAGAGACCCTCAC GCTGACCTGCACCGTCTCTGGGTTCTCACTCAGGAATGCTAGAATGGGTGTAAGCTG GATCCGTCAGCCTCCCGGGAAGGCCCTGGAGTGGCTTGCACACATTTTTTCGAATGA CGAAAAAACCTACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAGGGACACCT CCAAAGGCCAG GTGGTCCTTACCATGACCAAGATGGACCCTGTGGACACAGCCACA TATTACTGTGCACGGATACCCTACTATGGTTCGGGGAGTCATAACTACGGTATGGAC GTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEP ID NP:25)

Clone 2E7 NS AA (CDR underlined)

QVTLKESGPVLVKPTETLTLTCT VSGF SLRNARMGVSWIROPPGKALEWLAHIF SNDEK TYSTSLKSRLTISRDTSKGPVVLTMTKMDPVDTATYYCARIPYYGSGSHNYGMDVWGP GTTVTVSS (SEP ID NP:26)

Clone 2E7 NS AACDR1:

NARMGVS (SEP ID NP: 17)

Clone 2E7 NS AA CDR2: HIFSNDEKTYSTSLKS (SEQ ID NP:26)

Clone 2E7 NS AA CDR3: IPYYGSGSHNYGMDV (SEP ID NP:27)

Clone 2E7 LC DNA

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCAAGTCAGGACATTAGAAATGATTTCGGCTGGTATCAACA GAAACCAGGGAAAGCCCCTCAGCGCCTGCTCTATGCTGCATCCACTTTGCAAAGTGG GGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCA GCAGCCTGCAGC CTGAAGATTTTGCAACTTATTACTGTCTACAGTATAATACTTACC

CGTGGACGTTCGGTCAGGGAACGAAGGTGGAAATCAAACGA (SEP ID ND: 28)

Clone 2E7 LC AA (CDR underlined)

DIQMTOSPSSLSASVGDRVTITCRASQDIRNDFGWYOOKPGKAPORLLYAASTLOSGVP SRFSGSGSGTEFTLTISSLQPEDFATYYCLQYNTYPWTFGOGTKVEIKR (SEP ID ND: 29)

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Clone 2E7 LC AL CDR1: RASQDIRNDFG (SEQ ID NO: 30)

Clone 2E7 LC AA CDR2: AASTLQS (SEQ ID NO: 31)

Clone 2E7 LHC AA CDR3: LQYNTYPWT (SEQ ID NO: 32)

Clone 8B5 NS DNA

CAGATACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGTAGCGTCTGGATTCACCTTCAAGAACTATGGCATGCACTGGGTCCG CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGTATGATGGAAGTA ATGAATACTATGGAGACCCCGTGAAGGGCCGATTCACCATCTCCAGGACAACTCC AAGAACATGTTGTA TCTGCAAATGAACAGCCTGAGAGCCGATGACACGGCTGTGTA TTACTGTGCGAGGTCGGGAATAGCAGTGGCTGGGGCCTTTGACTACTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 33)

Clone 8B5 NS AA (CDRs are underlined)

OIOLVESGGGVVOPGRSLRLSCVASGFTFKNYGMHWVROAPGKGLEWVAVIWYDGSN EYYGDPVKGRFTISRDNSKNMLYLOMNSLRADDTAVYYCARSGIAVAGFDYWGOGT LVTVSS (SEQ Ш NO: 34)

Clone 8B5HC AACDR1: NYGMH (SEQ ID NO: 34)

Clone 8B5 NS AA CDR2: VIWYDGSNEYYGDPVKG (SEQ ID NO: 35)

Clone 8B5HC AACDR3: SGIAVAGAFDY (SEQ ID NO: 36)

Clone 8B5 LC DNA

GAAATTGTGTTGACGCAGTCTCCAGACACCCTGTCTTTGTCTCCAGGGGAAAAAGCC ACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTGGCCTGGTACCAG CAGAAACCTGGACAGGCTCCCAGTCTCCTCATCTATGTTGCATCCAGAAGGGCCGCT GGCATCCCTGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGG AGCCTGAAGATTTTGGAATGTTTTACTGTCAACACTATGGTAGGACA CCATTCACTTTCGGGCCCTGGGACCAAAGTGGATATCAAACGA (SEQ ID NO: 37)

Clone 8B5 LC AA (CDR underlined)

EIVLTOSPDTLSLSPGEKATLSCRASOSVSSSFLAWYOQKPGOAPSLLIYVASRRAAGIPD RFSGSGSGTDFTLTISRLEPEDFGMFYCQHYGRTPFTFGPGTKVDIKR (SEQ ID NO:41)

Clone 8B5 LC AA CDR1: RASQSVSSSFLA (SEQ Ш NO: 38)

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Clone 8B5 LC AL CDR2: VASRRAA (SEQ ID NO: 39)

Clone 8B5LC AACDR3: QHYGRTPFT (SEQ ID NO: 40)

Clone 4E9 NS DNA

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATACACTGGGTGCG ACAGGCCCCTGAACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTG GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGGCCAGGGACACGTCC ATCAGCAC AGTTTACATGGACCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTA TTACTGTGCGAGAATACCGGTGGTAACTCGGTCTTTGACTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCA (SEQ ID NO: 41)

Clone 4E9 NS AA (CDR underlined)

OVOLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVROAPEOGLEWMGWINPNSGG TNYAQKFQGRVTMARDTSISTVYMDLSRLRSDDTAVYYCARIRGGNSVFDYWGOGTL VTVSS (SEQ ID NO: 42)

Clone 4E9 NS AACDR1: GYYIH (SEQ ID NO: 43)

Clone 4E9 NS AA CDR2: WINPNSGGTNYAQKFQG (SEQ ID NO: 44)

Clone 4E9 NS AA CDR3: IRGGNSVFDY (SEQ ID NO: 45)

Clone 4E9 LC DNA

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCC ACCATCAACTGCAAGTCCACCCAGAGTATTTTATACACCTCCAACAATAAGAACTTC TTAGCTTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTCATTTCCTGGGCA TCTATCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGA TTTCGCTCTC ACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCA ACAATATTTTAGTACTATGTTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACG A (SEQ ID NO: 46)

Clone 4E9 LC AA (CDR underlined)

DIVMTOSPDSLAVSLGERATINCKSTQSILYTSNNKNFLAWYOOKPGOPPKLLISWASIR ESGVPDRFSGSGSGTDFALTISSLOAEDVAVYYCOQYFSTMFSFGOGTKLEIKR (SEQ ID NO: 47)

Clone 4E9 LC AA CDR1: KSTQSILYTSNNKNFLA (SEQ ID NO: 48)

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Clone 4E9 LC AL CDR2: WASIRES (SEQ Ш NO: 49)

Clone 4E9 LC AACDR3: QQYFSTMFS (SEQ ID NO: 50)

Clone 11F11 NS DNA

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTC CCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGTGCATACTACTGGACTTG GATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCCATTACAGTG GGAGCACCTACTCCAACCCGTCCCTCAAGAGTCGAATTACCATATCGTTAGACACGT CTAAGAACCAGTT CTCCCTGAAGCTGAACTCTGTGACTGCCGCGGACACGGCCGTGT ATTACTGTGCGAGACAAGAGGACTACGGTGGTTTGTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTTTCCTCA (SEQ ID NO: 51)

Clone 11F11 NS AA (CDRs are underlined)

OVOLQESGPGLVKPSOTLSLTCTVSGGSISSGAYYWTWIROHPGKGLEWIGYIHYSGST YSNPSLKSRITISLDTSKNOFSLKLNSVTAADTAVYYCARQEDYGGLFDYWGOGTLVTV SS (SEQ ID NO: 52)

Clone 11F11 HC AACDR1:

Clone 11F1 HC AACDR2:

Clone 11F1 HC AACDR3:

SGAYYWT (SEQ ID NO: 53)

YIHYSGSTYSNPSLKS (SEQ Ш NO: 54)

QEDYGGLFDY (SEQ ID NO: 55)

Clone 11F11 LC DNA

GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAAT CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCACCGACTTAGCCTGGTACCAGCA GATGCCTGGACAGGCTCCCCGGCTCCTCATCTATGATGCTTCCACCAGGGCCACTGG TTTCCCAGCCAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCACGCTCACCATCAG CAGCCTGCAGGCTG AAGATTTTGCAGTTTATTACTGTCAACATTATAAAACCTGGCC

TCTCACTTTCGGCGGAGGGACTAAGGTGGAGATCAAACGA (SEQ ID NO: 56)

Clone 11F11 LC AA (CDR underlined)

EIVMTOSPATLSVSPGERITLSCRASOSVTTDLAWYOOMPGOAPRLLIYDASTRATGFPA RFSGSGSGTDFTLTISSLOAEDFAVYYCQHYKTWPLTFGGGTKVEIKR (SEQ ID NO: 57)

Clone 11F11 LC AACDR1: RASQSVTTDLA (SEQ ID NO: 58)

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Clone 11F1 LCAACDR2: DASTRAT (SEQ GO NO: 59)

Clone 11F1 LC AACDR3: QHYKTWPLT (SEQ ID NO: 60)

10E3 CD28 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTGACCCTCAAAGAGTCTGGACCCGTGCTCGTAAAACCTACGGA GACCCTGACACTCACCTGCACAGTCTCCGGCTTCAGCCTCATCAATGCCAGGATGG AGTTTCCTGGATCAGGCAACCGCCCGGAAAGGCCCTGGAATGGCTCGCACATATTTT CAGTAACGCTGAAAAAAGC TATCGGACTTCTCTGAAAAGTCGGCTCACGATTAGTA AGGACACATCCAAGAGCCAAGTGGTGCTTACGATGACTAACATGGACCCTGTGGAT ACTGCAACCTATTACTGTGCTCGAATCCCTGGTTATGGCGGAAATGGGGACTACCAC TACTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTTACTGTTTCAAGCGGAGG GGGAGGGAGTGGGGGTGGCGGATCTGGCGGAGG AGGCAGCGATATCCAGATGACG CAGTCCCCTAGTTCACTTTCCGCATCCCTGGGGGATCGGGTTACCATTACATGCCGC GCGTCACAGGGTATCCGGAATGATCTGGGATGGTACCAGCAGAAGCCGGGAAAGGC TCCTAAGCGCCTCATCTACGCCAGCTCCACCCTGCAGAGTGGAGTGCCCTCCCGGTT TTCAGGCAGTGGCTCCGGTACGGAGTTTACTCTTACAATTAGCAG CCTGCAGCCAGA AGATTTTGCAACTTACTACTGTTTGCAGCATAATAATTTCCCCTGGACCTTTGGTCAG GGCACCAAGGTGGAGATCAAAAGAGCAGCCGCCATCGAAGTAATGTATCCCCCCC GTACCTTGACAATGAGAAGTCAAATGGAACCATTATCCATGTTAAGGGCAAACACC TCTGCCCTTCTCCACTGTTCCCTGGCCCTAGTAAGCCGTTTTGGGTGCTGGTGGTAG T CGGTGGGGTGCTGGCTTGTTACTCTCTTCTCGTGACCGTCGCCTTTATAATCTTTTGG GTCAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACG CCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGC TGCCTATCGGAGCCGAGTGAAATTTTTCTAGATCAGCTGATGCTCCCGCCTATCAGCA GGGACAGAATCAACT TTACAATGAGCTGAACCTGGGTCGCAGAGAAGAGTACGACG TTTTGGACAAACGCCGGGGCCGAGATCCTGAGATGGGGGGGAAGCCGAGAAGGAA GAATCCTCAAGAAGGCCTGTACAACGAGCTTCAAAAAGACAAAATGGCTGAGGCGT ACTCTGAGATCGGCATGAAGGGCGAGCGGAGACGAGGCAAGGGTCACGATGGCTTG TATCAGGGCCTGAGTACAGCCACAAAGGA CACCTATGACGCCCTCCACATGCAGGC ACTGCCCCCACGCTAG (SEQ GO NO: 61)

Construct 10E3 CD28 AA (signal sequence in bold; CDRs underlined)

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MALPVTALLLPLALLLHAARPOVTLKESGPVLVKPTETLTLTCTVSGFSLINARMGVS WIRQPPGKALEWLAHIFSNAEKSYRTSLKSRLTISKDTSKSQVVLMTTNMDPVDTATYY CARIPGYGGNGDYHYYGMDVWGOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSL SASLGDRVTITCRASQGIRNDLGWYOOKPGK APKRLIYASSTLOSGVPSRFSGSGSGTEF TLTISSLOPEDFATYYCLQHNNFPWTFGOGTKVEIKRAAAAIEVMYPPPYLDNEKSNGTII HVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQG QNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ Ш NO: 62)

10E3 CD28T DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAAGTTACTTTGAAGGAGTCTGGACCTGTACTGGTGAAGCCAACCGA GACACTGACACTCACGTGTACAGTGAGTGGTTTTTCCTTGATCAACGCAAGGATGGG CGTCAGCTGGATCAGGCAACCCCCTGGCAAGGCTCTGGAATGGCTCGCTCACATATT CAGCAATGCCGAAA AAAGCTACCGGACAAGCCTGAAATCCCGCCTGACTATTTCCA AGGACACTTCTAAGTCTCAGGTGGTGCTGACCATGACCAACATGGACCCGGTGGAC ACCGCCACCTATTACTGCGCAAGAATCCCTGGGTATGGTGGGAATGGTGACTACCAT TATTATGGGATGGATGTGTGGGGGCAAGGCACAACCGTAACGGTCTCAAGCGGTGG GGGAGGCTCAGGGGGCGGAGGCTCCGGA GGTGGCGGCTCCGACATTCAGATGACCC AAAGCCCGTCCAGCCTGTCCGCCAGCCTGGGAGATAGAGTGACAATCACGTGTAGA GCTTCCCAAGGGATAAGAAATGATCTCGGGTGGTATCAGCAGAAGCCCGGCAAAGC CCCCAAAAGGCTTATATATGCTAGTAGTACACTGCAGTCTGGAGTTCCTTCCCGATT TTCAGGTAGCGGCTCCGGTACAGAGTTCACCCTCACGATAAG CTCACTCCAGCCTGA GGATTTCGCAACGTACTACTGCCTCCAGCACAACAATTTTCCCTGGACTTTCGGCCA GGGCACCAAGGTGGAGATCAAGAGGGCCGCTGCCCTTGATAATGAAAAGTCAAACG GAACAATCATTCACGTGAAGGGCAAGCACCTCTGTCCGTCACCCTTGTTCCCTGGTC CATCCAAGCCATTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCT CT GCTCGTCACCGTGGCTTTTATAATCTTCTGGGTTAGATCCAAAAGAAGCCGCCTGCT CCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACT ACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCCGAGTGAAATTTT CTAGATCAGCTGATGCTCCCGCCTATCAGCAGGGACAGAATCAACTTTACAATGAGC TGAACCTGGGTCGCAGAGA AGAGTACGACGTTTTGGACAAACGCCGGGGCCGAGAT CCTGAGATGGGGGGGAAGCCGAGAAGGAAGAATCCTCAAGAAGGCCTGTACAACG

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AGCTTCAAAAAGACAAAATGGCTGAGGCGTACTCTGAGATCGGCATGAAGGGCGAG CGGAGACGAGGCAAGGGTCACGATGGCTTGTATCAGGGCCTGAGTACAGCCACAAA GGACACCTATGACGCCCTCCACATGCAGGCACTGCCCCACGCTAG (SEQ ID NO: 63)

10E3 CD28T AA design (signal sequence in bold; CDRs underlined)

MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGFSLINARMGVS WIROPPGKALEWLAHIFSNAEKSYRTSLKSRLTISKDTSKSOVVLTMTNMDPVDTATYY CARIPGYGGNGDYHYYGMDVWGOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSL SASLGDRVTITCRASQGIRNDLGWYOOKPGKAP KRLIYASSTLOSGVPSRFSGSGSGTEF TLTISSLOPEDFATYYCLQHNNFPWTFGOGTKVEIKRAAALDNEKSNGTIIHVKGKHLCP SPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO: 64)

10E3 CD8 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTGACACTCAAGGAATCAGGGCCCGTACTGGTGAAACCTACTG AGACCCTGACACTGACTTGCACCGTGTCTGGGTTCTCTCTGATTAACGCTCGAATGG

GTGTGAGTTGGATACGCCAGCCTCCAGGGAAGGCTCTGGAGTGGTTGGCCCACATTT TCTCCAACGCCGAGAAGAGCTACAGGACTAGTCTGAAGTCCAGACTTACCATTTCCA AAGACACAAGTAAATCACAGGTGGTGCTGACAATGACAAACATGGACCCGGTTGAT ACTGCTACCTATTATTGTGCCCGCATTCCCGGCTACGGCGGCAATGGCGACTATCAC TATTAT GGTATGGATGTCTGGGGGCAGGGGACCACTGTTACCGTGTCCAGCGGGGGT GGTGGCAGCGGAGGTGGAGGGAGCGGTGGTGGGGGGAGTGATATTCAGATGACCC AGAGCCCTAGCTCTCTTTCCGCTTCTCTGGGCGATAGAGTCACCATCACCTGCCGGG CCTCTCAAGGCATCCGGAACGATCTTGGATGGTATCAGCAGAAGCCCGGCAAGGCA CCAAAAAGGCTGATCTACGCAT CAAGCACCCTGCAATCTGGGGTGCCGTCCCGGTTT TCTGGTTCTGGTAGTGGGACCGAGTTTACTCTGACTATTTCTTCCCTGCAGCCTGAGG ACTTTGCTACGTACTATTGTCTGCAGCATAACAACTTCCCCTGGACGTTCGGGCAGG GTACGAAAGTGGAAATTAAGCGCGCCGCCGCCCTGTCCAACTCCATTATGTATTTCT CTCATTTTGTCCCAGTGTTCCTGCCCGCTAAA CCCACAACTACTCCGGCGCCCCGAC CGCCAACTCCCGCACCTACCATCGCAAGCCAGCCATTGAGCCTCCGACCTGAGGCAT GTAGACCAGCAGCCGGCGGTGCCGTGCACACAAGGGGACTGGATTTCGCCTGCGAC

- 38 044866

ATATATATTTGGGCCCCTCTGGCTGGAACCTGTGGGGTTCTGCTGCTCTCTCTCGTTA TTACACTGTATTGCAATCATCGCAATAGATCCAAAAGAAGCCGCCTGCTCCATAGCG ATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTT ACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCCGAGTGAAATTTTTCTAGATCAG CTGATGCTCCCGCC TATCAGCAGGGACAGAATCAACTTTACAATGAGCTGAACCTGG GTCGCAGAGAAGAGTACGACGTTTTGGACAAACGCCGGGGCCGAGATCCTGAGATG GGGGGGAAGCCGAGAAGGAAGAATCCTCAAGAAGGCCTGTACAACGAGCTTCAAA AAGACAAAATGGCTGAGGCGTACTCTGAGATCGGCATGAAGGGCGAGCGGAGACG AGGCAAGGGTCACGATGGCTTGTATCAGGGCCTGAG TACAGCCACAAAGGACACCT ATGACGCCCTCCACATGCAGGCACTGCCCCACGCTAG (SEQ ID NO: 65)

Design 10E3 CD8 AA (signal sequence in bold; CDRs underlined) MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLTLTCTVSGFSLINARMGVS WIROPPGKALEWLAHIFSNAEKSYRTSLKSRLTISKDTSKSOVVLTMTNMDPVDTATYY CARIPGYGGNGDYHYYGMDVWGOGTTVTVSSGGGG SGGGGSGGGGSDIOMTOSPSSL SASLGDRVTITCRASQGIRNDLGWYOOKPGKAPKRLIYASSTLOSGVPSRFSGSGSGTEF TLTISSLOPEDFATYYCLQHNNFPWTFGOGTKVEIKRAAALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL SLVITLY CNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 66)

8B5 CD28 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGATCCAGTTGGTGGAATCAGGGGGCGGTGTGGTGCAGCCGGGTA GGAGCCTGAGACTGTCATGCGTGGCGTCTGGCTTCACATTCAAGAACTACGGCATGC ACTGGGTGCGACAGGCCCCCGGAAAGGGTTTGGAGTGGGTCGCCGTGATCTGGTAC GACGGATCTAATGAG TATTACGGAGATCCTGTGAAGGGAAGGTTCACCATCTCCCG CGACAATAGCAAAAATATGCTCTACCTGCAAATGAACTCACTCAGGGCGGATGATA CGGCGGTCTACTATTGCGCTCGCTCAGGGATTGCTGTGGCCGGCGCATTCGATTACT GGGGACAGGGTACCCTGGTGACAGTATCAAGCGGAGGCGGCGGCTCTGGCGGCGGC GGATCTGGCGGGGGGGGAAGTGAGATTGTGTT GACACAGTCTCCCGATACCCTGTC

- 39 044866

ACTGTCACCCGGCGAGAAGGCAACGCTGAGTTGCAGAGCAAGCCAGTCAGTCTCCT CTTCTTTTCTGGCCTGGTATCAGCAAAAACCAGGTCAGGCACCATCTCTCCTGATTTA CGTTGCCAGCAGACGGGCGGCTGGCATTCCCGACAGGTTCTCTGGAAGCGGATCTG GGACCGATTTTACCCTGACAATTAGCCGCTTGGAGCCCGAAGACTTTGGTATGTTTT ACTGCCAGCACTAC GGAAGGACACCTTTTCACATTTGGCCCGGGCACGAAAGTCGAT ATAAAACGCGCAGCCGCCATTGAAGTAATGTACCCACCACCTTATTTGGACAATGA AAAGTCCAATGGTACCATTATTCACGTCAAGGGAAAGCATCTCTGTCCAAGCCCTCT GTTCCCCGGCCCCTCCAAACCATTCTGGGTGCTGGTGGTCGTCGGCGGAGTTCTGGC CTGCTATTCTCTGCTCGTGACTGTTG CATTCATCATTTTCTGGGTGAGATCCAAAAGA AGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACA AGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCCG AGTGAAATTTTTCTAGATCAGCTGATGCTCCCGCCTATCAGCAGGGACAGAATCAACT TTACAATGAGCTGAACCTGGGTCGCAGAAGAGTACGACGTT TTGGACAAACGCC GGGGCCGAGATCCTGAGATGGGGGGGAAGCCGAGAAGGAAGAATCCTCAAGAAGG CCTGTACAACGAGCTTCAAAAAGACAAAATGGCTGAGGCGTACTCTGAGATCGGCA TGAAGGGCGAGCGGAGACGAGGCAAGGGTCACGATGGCTTGTATCAGGGCCTGAGT ACAGCCACAAAGGACACCTATGACGCCCTCCACATGCAGGCACTGCCCCCACGCTA G (SEQ ID NO: 67)

8B5 CD28 AA design (signal sequence in bold)

MALPVTALLLPLALLLHAARPQIQLVESGGGVVQPGRSLRLSCVASGFTFKNYGMHW VRQAPGKGLEWVAVIWYDGSNEYYGDPVKGRFTISRDNSKNMLYLQMNSLRADDTAV YYCARSGIAVAGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPDTLSLSPGE KATLSCRASQSVSSSFLAWYQQKPG QAPSLLIYVASRRAAGIPDRFSGSGSGTDFTLTISR LEPEDFGMFYCQHYGRTPFTFGPGTKVDIKRAAAAIEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ GO NO: 68)

8B5 CD28T DNA design (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGATTCAGCTCGTGGAGTCAGGTGGTGGCGTGGTTCAGCCCGGACG GTCCCTGCGACTCTCTTGTGTGGCAAGCGGATTTACCTTTAAGAACTATGGCATGCA

- 40 044866

CTGGGTGAGGCAGGCCCCTGGAAAAGGACTGGAGTGGGTTGCTGTGATCTGGTACG ACGGGTCCAACGAATATTATGGCGATCCTGTGAAGGGACGGTTTACAATCTCACGC GATAACTCAAAGAACATGCTGTACCTGCAAATGAACTCTCTGCGCGCTGATGACACT GCCGTGTATTATTGCGCTCGGAGTGGTATCGCCGTCGCAGGAGCATTTGATTATTGG GGGCAAGGGA CCCTCGTGACAGTGAGTTCCGGAGGGGGAGGTTCTGGTGGAGGCGG CTCTGGTGGGGGAGGCAGCGAGATCGTTCTGACCCAGTCTCCTGACACACTGTCACT GTCCCCTGGTGAAAAGGCCACACTGTCTTGTAGAGCGTCCCAGAGCGTTTCCAGTTC CTTCCTTGCATGGTATCAACAAAAACCCGGGCAGGCTCCAAGCTTGCTGATCTACGT GGCCAGCCGCCGGGCCG CAGGCATCCCTGATAGGTTTAGCGGTTCTGGGAGCGGGA CGGACTTCACCTTGACAATATCACGGCTGGAACCCGAAGACTTCGGAATGTTTTATT GCCAGCACTACGGAAGAACTCCATTCACCTTTGGCCCGGGAACGAAGGTAGACATC AAGAGAGCAGCAGCCCTCGACAACGAGAAATCCAATGGAACCATTATCCATGTGAA GGGGAAACATCTCTGCCCTTCACCATTGTTCCCTGGAC CCAGCAAGCCTTTTTGGGT TCTGGTCGTGGTGGGGGGCGTCCTGGCTTGTTACTCCCTCCTCGTTACAGTCGCCTTC ATAATCTTTTGGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAAT ATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCT AGAGATTTCGCTGCCTATCGGAGCCGAGTGAAATTTTCTAGATCAGCTGAT GCTCCC GCCTATCAGCAGGGACAGAATCAACTTTACAATGAGCTGAACCTGGGTCGCAGAGA AGAGTACGACGTTTTGGACAAACGCCGGGGCCGAGATCCTGAGATGGGGGGGAAGC CGAGAAGGAAGAATCCTCAAGAAGGCCTGTACAACGAGCTTCAAAAAGACAAAAT GGCTGAGGCGTACTCTGAGATCGGCATGAAGGGCGAGCGGAGACGAGGCAAGGGT CACGATGGCTTG TATCAGGGCCTGAGTACAGCCACAAAGGACACCTATGACGCCCT CCACATGCAGGCACTGCCCCCACGCTAG (SEQ ID NO: 69)

8B5 CD28T AA design (signal sequence in bold)

MALPVTALLLPLALLLHAARPQIQLVESGGGVVQPGRSLRLSCVASGFTFKNYGMHW VRQAPGKGLEWVAVIWYDGSNEYYGDPVKGRFTISRDNSKNMLYLQMNSLRADDTAV YYCARSGIAVAGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPDTLSLSPGE KATLSCRASQSVSSSFLAWYQQKPG QAPSLLIYVASRRAAGIPDRFSGSGSGTDFTLTISR LEPEDFGMFYCQHYGRTPFTFGPGTKVDIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPG PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ Ш NO: 70)

- 41 044866

8B5 CD8 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGATACAGCTTGTCGAATCCGGTGGCGGGGTGGTGCAGCCTGGAC GCAGCCTGCGGCTTTCTTGCGTGGCCAGCGGATTTACCTTCAAGAACTACGGGATGC ATTGGGTCCGCCAGCACCCGGCAAAGGCCTTGAGTGGGTTGCAGTGATCTGGTAC GACGGCAGTAACGA GTATTATGGCGACCCCGTGAAGGGAAGGTTTACTATTTCAAG AGATAATAGTAAGAACATGTTGTATCTGCAAATGAACAGTCTGAGAGCGGACGACA CTGCCGTGTACTACTGTGCTCGCTCCGGCATCGCTGTGGCAGGGGCCTTTGACTACT GGGGTCAGGGGACGCTGGTCACGGTTAGTTCCGGGGGCGGTGGTTCCGGAGGAGGC GGTTCCGGCGGCGGCGGATCAG AAATCGTTCTTACTCAGAGTCCCGATACGCTGTCC TTGTCTCCGGGAGAAAAAGCCACACTGAGCTGCCGAGCCTCACAGTCAGTAAGTTCT TCATTCCTCGCCTGGTACCAGCAAAAACCGGGCAGGCCCCTTCCCTGCTTATCTAC GTGGCCTCTAGGAGAGCCGCCGGTATTCCTGACCGGTTCAGCGGAAGTGGTTCCGG GACTGATTTTACGCTCACGATCTCCCGATTGG AGCCCGAGGATTTCGGGATGTTCTA CTGTCAGCATTATGGAAGAACGCCCTTTACCTTCGGTCCGGGAACTAAGGTTGATAT TAAGCGGGCTGCTGCCCTTAGCAACTCCATCATGTATTTTTCTCACTTCGTGCCAGTA TTCCTGCCAGCCAAACCGACCACAACCCCAGCACCTAGACCTCCTACTCCGGCTCCC ACCATAGCTTCACAGCCGCTGAGTTTGAGGCCAGAGGCCTGTCGGCC TGCTGCAGGC GGAGCAGTTCACACCAGGGGACTTGACTTTGCATGTGACATCTATATTTGGGCTCCA CTGGCGGGAACCTGCGGGGTGCTCCTTTTGTCACTCGTTATCAACTGTATTGCAAT CATAGGAATAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGAC TCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAG ATTTCGCTGC CTATCGGAGCCGAGTGAAATTTTCTAGATCAGCTGATGCTCCCGCCT ATCAGCAGGGACAGAATCAACTTTACAATGAGCTGAACCTGGGTCGCAGAGAAGAG TACGACGTTTTGGACAAACGCCGGGGCCGAGATCCTGAGATGGGGGGGAAGCCGAG AAGGAAGAATCCTCAAGAAGGCCTGTACAACGAGCTTCAAAAAGACAAAATGGCTG AGGCGTACTCTGAGATCGGCATGAAGGGC GAGCGGAGACGAGGCAAGGGTCACGA TGGCTTGTATCAGGGCCTGAGTACAGCCACAAAGGACACCTATGACGCCCTCCACAT GCAGGCACTGCCCCCACGCTAG (SEQ Ш NO: 71)

Design 8B5 CD8 AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQIQLVESGGGVVQPGRSLRLSCVASGFTFKNYGMHW VRQAPGKGLEWVAVIWYDGSNEYYGDPVKGRFTISRDNSKNMLYLQMNSLRADDTAV

- 42 044866

YYCARSGIAVAGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPDTLSLSPGE KATLSCRASQSVSSSFLAWYQQKPGQAPSLLIYVASRRAAGIPDRFSGSGSGTDFTLTISR LEPEDFGMFYCQHYGRTPFTFGPGTKVDIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAP RPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (SEQ ID NO: 72)

4E9 CD28 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTGCAGCTGGTGCAGAGTGGGGCAGAAGTAAAGAAGCCTGGTG CCTCTGTCAAAGTTAGTTGCAAAGCATCTGGGTATACTTTCACCGGTTACTATATCC ATTGGGTTCGGCAGGCCCCGGAGCAGGGACTGGAGTGGATGGGCTGGATCAACCCA AATTCAGGCGGCACTA ACTATGCTCAAAAGTTCCAGGGCAGGGTCACAATGGCCCG GGATACTTCAATTAGCACCGTCTATATGGATCTTAGTCGGCTGCGCAGTGACGATAC CGCTGTCTACTATTGCGCAAGGATCAGGGGCGGCAATTCTGTTTTTGACTATTGGGG CCAGGGAACACTGGTGACCGTCTCCTCTGGTGGAGGCGGTAGTGGTGGAGGCGGGT CCGGAGGAGGGGGCTCCGATATAGTGAT GACTCAAAGTCCCGATAGCTTGGCAGTA TCTCTTGGGGAACGCGCCACTATTAACTGTAAATCCACCCAGTCCATTCTCTATACCT CTAACAACAAGAATTTCCTCGTGGTATCAGCAAAAACCCGGGCAGCCACCTAAA CTGCTTATATCCTGGGCCAGCATCAGGGAGTCCGGCGTCCCTGATCGGTTCAGCGGT AGTGGCAGCGGGACAGACTTCGCTCTGACCATCAGTA GCCTCCAGGCTGAAGATGT CGCAGTGTATTATTGCCAGCAGTACTTCAGCACGATGTTTAGCTTCGGGCAGGGAAC CAAGCTGGAAATAAAGAGAGCTGCAGCAATCGAGGTGATGTACCCACCTCCATATC TGGACAATGAAAAGTCCAATGGCACTATCATACACGTGAAGGGCAAACACCTGTGT CCATCTCCACTTTTCCCGGGCCCGTCTAAACCTTTCTGGGTGCTGGTGGTGG TGGGC GGAGTTCTGGCCTGTTATTCACTGCTGGTCACCGTGGCTTTCATCATTTTTTGGGTAA GATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGC CCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCC TATCGGAGCCGAGTGAAATTTTTCTAGATCAGCTGATGCTCCCGCCTATCAGCAGGGA CAGAATCAACT TTACAATGAGCTGAACCTGGGTCGCAGAGAAGAGTACGACGTTTT GGACAAACGCCGGGGCCGAGATCCTGAGATGGGGGGGAAGCCGAGAAGGAAGAAT CCTCAAGAAGGCCTGTACAACGAGCTTCAAAAAGACAAAATGGCTGAGGCGTACTC TGAGATCGGCATGAAGGGCGAGCGGAGACGAGGCAAGGGTCACGATGGCTTGTATC

- 43 044866

AGGGCCTGAGTACAGCCACAAAGGACACCTATGACGCCCTCCACATGCAGGCACTG CCCCCACGCTAG (SEQ ID NO: 73)

Design 4E9 CD28 AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHW VRQAPEQGLEWMGWINPNSGGTNYAQKFQGRVTMARDTSISTVYMDLSRLRSDDTAV YYCARIRGGNSVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGE RATINCKSTQSILYTSNNKNFLAWY QQKPGQPPKLLISWASIRESGVPDRFSGSGSGTDF ALTISSLQAEDVAVYYCQQYFSTMFSFGQGTKLEIKRAAAAIEVMYPPPYLDNEKSNGTII HVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 74)

4E9 CD28T DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTACAGCTGGTGCAGAGCGGGGCCGAGGTCAAAAGCCCGGGG CTTCAGTTAAGGTTAGCTGCAAGGCTTCCGGCTACACCTTTACCGGTTACTATATTCA CTGGGTTAGACAGGCACCTGAGCAAGGACTGGAGTGGATGGGGTGGATTAACCCCA ATAGCGGTGGGACCAACT ACGCCCAGAAGTTTCAAGGCCGAGTGACAATGGCACGA GACACCTCCATTTCCACTGTGTACATGGACTTGAGCCGCCTCAGGTCAGACGACACC GCAGTGTACTACTGTGCGCGAATCCGCGGCGGAAACAGCGTGTTTGACTACTGGGG TCAGGGCACGTTGGTGACCGTGTCTTCCGGAGGGGGGGGATCTGGTGGCGGGGGCT CCGGCGGAGGCGGTAGTGATATTGTGATGACT CAGTCACCGGACAGTCTTGCTGTTT CACTTGGTGAGAGGGCCACCATAAATTGTAAAAGCACCCAGAGCATTCTCTACACA TCTAACAACAAAAATTTCCTGGCCTGGTACCAGCAGAAGCCCGGACAGCCACCCAA ATTGCTGATTAGCTGGGCCAGCATTCGAGAATCTGGGGTTCCGGACCGCTTTTCCGG GTCTGGCTCTGGGACCGACTTCGCTTTGACCATAAGCTCTCTTCAGGC CGAAGACGT CGCAGTATACTATTGTCAACAGTATTTTTCTACCATGTTTTCCTTCGGCCAGGGAACT AAGTTGGAGATCAAGAGAGCAGCTGCATTGGATAATGAGAAGTCCAATGGCACTAT TATCCACGTGAAAGGTAAACACCTGTGTCCCTCACCCCTGTTTCCAGGACCTAGTAA ACCATTCTGGGTCTTGGTTGTAGTCGGGGGCGTTTTGGCATGTTATTCCCTTCTTGTG ACAGTCGCCTTTATCATTTTCTGGGTGAGATCCAAAAGAAGCCGCCTGCTCCATAGC GATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCC

- 44 044866

TTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCCGAGTGAAATTTTCTAGATC AGCTGATGCTCCCGCCTATCAGCAGGGACAGAATCAACTTTACAATGAGCTGAACCT GGGTCGCAGAGAAGAGTACGACGTTTTGGACAAACGCCGGGGCCGAGATCCTGAGA TGGGGGGGAAGCCGAGAAGGAAGAATCCTCAAGAAGGCCTGTACAACGAGCTTCA AAAAGACAAAATGG CTGAGGCGTACTCTGAGATCGGCATGAAGGGCGAGCGGAGA CGAGGCAAGGGTCACGATGGCTTGTATCAGGGCCTGAGTACAGCCACAAAGGACAC CTATGACGCCCTCCACATGCAGGCACTGCCCCACGCTAG (SEQ ID NO: 75)

Design 4E9 CD28T AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHW VRQAPEQGLEWMGWINPNSGGTNYAQKFQGRVTMARDTSISTVYMDLSRLRSDDTAV YYCARIRGGNSVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGE RATINCKSTQSILYTSNNKNFLAWY QQKPGQPPKLLISWASIRESGVPDRFSGSGSGTDF ALTISSLQAEDVAVYYCQQYFSTMFSFGQGTKLEIKRAAALDNEKSNGTIIHVKGKHLCP SPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO: 76)

4E9 CD8 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAAGTTCAGCTTGTGCAGAGCGGAGCTGAGGTGAAAAAACCAGGCG CCTCCGTTAAGGTGTCTTGCAAAGCCAGCGGATACACATTTACCGGGTACTATATTC ACTGGGTGAGGCAGGCCCCTGAACAGGGCCTTGAATGGATGGGGTGGATCAATCCA AATTCCGGGGGAACCAATTATGCTCAGAAATTTCAGGGCAGAGTGACAATGGCCAG GGACACCTCAATCAGCACAGTCTACATGGACCTGAGCCGCCTGAGGTCTGATGACA CAGCCGTCTACTACTGTGCCCGGATCAGAGGGGGAAACAGTGTCTTCGACTATTGGG GGCAGGGAACCCTGGTGACTGTCTCCTCCGGGGGAGGGGGTAGCGGGGGAGGCGGC AGCGGCGGGGGTGGTTCTGACATTGTTATGACCCAATCCCCAGACTCTCTGGCCGTG AGCCTGGGTGAGAGAGCCACCATCAATTGCAAGTCCACCCAGAGCATACTCTATAC GTCAAACAATAAGAATTTCCTGGCGTGGTATCAGCAAAAGCCGGGTCAACCACCCA AGTTGTTGATTAGCTGGGCATCAATTCGAGAATCTGGCGTCCCTGATAGGTTTAGCG GGAGCGGTAGTGGAACCGACTTTGCGCTGACCATTTCATCCCTTCAGGCAGAGGAC

- 45 044866

GTGGCTGTGTATTACTGTCAACAGTACTTCAGCACGATGTTTTCTTTCGGCCAGGGG ACGAAGCTGGAGATAAAGCGGGCCGCAGCACTCAGCAACAGCATCATGTACTTTTC TCATTTCGTCCCAGTTTTTCTCCCCGCCAAACCCACCACTACCCCTGCTCCTAGGCCT CCCACTCCCGCACCCACCATTGCTTCCCAACCTCTGTCATTGAGGCCCGAAGCCTGC AGACCTGCCGCAGGA GGGGCTGTGCACACCCGCGTCTGGATTTTGCTTGTGATATC TACATTTGGGCCCCTTTGGCCGGAACCTGCGGAGTGTTGTTGCTGAGCCTTGTTATC ACGTTGTACTGTAATCACAGAAACAGATCCAAAAGAAGCCGCCTGCTCCATAGCGA TTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTA CGCACCACCTAGAGATTTCGCTGCCTA TCGGAGCCGAGTGAAATTTTCTAGATCAGC TGATGCTCCCGCCTATCAGCAGGGACAGAATCAACTTTACAATGAGCTGAACCTGG GTCGCAGAGAAGAGTACGACGTTTTGGACAAACGCCGGGGCCGAGATCCTGAGATG GGGGGGAAGCCGAGAAGGAAGAATCCTCAAGAAGGCCTGTACAACGAGCTTCAAA AAGACAAAATGGCTGAGGCGTACTCTGAGATCGGCATGAAGGGCGAGCG GAGACG AGGCAAGGGTCACGATGGCTTGTATCAGGGCCTGAGTACAGCCACAAAGGACACCT ATGACGCCCTCCACATGCAGGCACTGCCCCCACGCTAG (SEQ ID NO: 77)

Construction 4E9 CD 8 AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHW VRQAPEQGLEWMGWINPNSGGTNYAQKFQGRVTMARDTSISTVYMDLSRLRSDDTAV YYCARIRGGNSVFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGE RATINCKSTQSILYTSNNKNFLAWY QQKPGQPPKLLISWASIRESGVPDRFSGSGSGTDF ALTISSLQAEDVAVYYCQQYFSTMFSFGQGTKLEIKRAAALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL SLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 78)

11F11 CD28 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTGCAGCTCCAAGAGTCAGGACCAGGACTTGTCAAACCAAGCC AGACCCTCAGCCTTACCTGCACCGTCAGCGGGGGCTCCATCAGCTCTGGGGCTTACT ACTGGACATGGATACGACAGCATCCCGGTAAAGGTCTGGAGTGGATCGGGTACATA

- 46 044866

CACTATAGTGGTTCCACATATTCTAATCCATCTCTTAAGAGTCGAATTACAATTTCAC TCGATACTTCAAAGAATCAGTTCAGCTTGAAACTGAACTCCGTGACCGCGGCTGACA CCGCCGTGTACTACTGTGCACGCCAAGAGGATTATGGCGGACTGTTCGATTATTGGG GGCAGGGAACTCTCGTGACAGTGAGCTCCGGCGGGGGCGGCAGCGGTGGGGGTGG AAGTGGTGGA GGGGGCAGCGAGATCGTGATGACCCAGAGTCCTGCCACACTGTCAG TGAGTCCTGGGGAGCGAATCACACTTTCCTGTCGAGCGTCTCAGTCCGTGACCACGG ACCTGGCGTGGTACCAGCAGATGCCAGCCAGGCGCCAAGACTCCTGATCTACGAC GCTTCTACCCGCGCTACTGGTTTCCCCGCCAGATTCTCCGGAAGCGGGTCCGGGACG GATTTTACACTTACCATCTCT TCATTGCAGGCTGAGGATTTTGCCGTGTACTACTGTC AGCATTACAAAACCTGGCCCCTCACTTTCGGGGGCGGAACAAAAGTGGAAATTAAA CGGGCAGCAGCTATTGAGGTGATGTACCCACCCCCCTACCTGGACAACGAGAAATC CAATGGCACCATCATCCACGTTAAGGGTAAGCACTTGTGTCCCTCACCACTCTTCCC TGGGCCTAGCAAGCCATTCTGGGTCCTGGTGGTCG TGGGAGGCGTGCTGGCCTGCTA TTCCCTCCTGGTTACCGTTGCCTTTATCATATTTTGGGTCAGATCCAAAAGAAGCCGC CTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAA CACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGCCGAGTGAAA TTTTCTAGATCAGCTGATGCTCCCGCCTATCAGCAGGGACAGAATCAACTTTA CAAT GAGCTGAACCTGGGTCGCAGAGAAGAGTACGACGTTTTGGACAAACGCCGGGGCCG AGATCCTGAGATGGGGGGGAAGCCGAGAAGGAAGAATCCTCAAGAAGGCCTGTAC AACGAGCTTCAAAAAGACAAAATGGCTGAGGCGTACTCTGAGATCGGCATGAAGGG CGAGCGGAGACGAGGCAAGGGTCACGATGGCTTGTATCAGGGCCTGAGTACAGCCA CAAAGGACACC TATGACGCCCTCCACATGCAGGCACTGCCCCACGCTAG (SEQ ID NO: 79)

Design 11F11 CD28 AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGAYYWT WIRQHPGKGLEWIGYIHYSGSTYSNPSLKSRITISLDTSKNQFSLKLNSVTAADTAVYYC ARQED YGGLFDYWGQGTLVTVS SGGGGSGGGGSGGGGSEIVMTQ SPATES VSPGERITL SCRASQSVTTDLAWYQQMP GQAPRLLIYDASTRATGFPARFSGSGSGTDFTLTISSLQAE DFAVYYCQHYKTWPLTFGGGTKVEIKRAAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCP SPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO: 80)

- 47 044866

11F11 CD28T DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTGCAGTTGCAGGAGAGCGGGCCAGGCCTGGTGAAGCCCAGCC AAACACTGAGCCTCACCTGTACTGTGTCCGGTGGTAGCATTTCCAGCGGGGCGTATT ATTGGACATGGATACGCCAACACCCTGGAAAAGGGTTGGAGTGGATTGGATACATC CATTATTCTGGGTCCAC CTATAGTAACCCTTCTCTCAAGTCTCGCATTACTATTAGTT TGGATACCTCTAAGAATCAGTTTAGTCTGAAGCTGAACAGTGTAACCGCCGCCGACA CCGCGGTCTACTACTGTGCTAGGCAGGAGGATTACGGGGGACTGTTCGATTACTGGG GCCAGGGGACATTGGTCACCGTTTCAAGCGGGGGCGGCGGATCTGGCGGAGGGGGA TCTGGAGGCGGAGGCTCTGAGATCGTA ATGACTCAGAGCCCAGCCACCCTGTCCGTC TCTCCCGGCGAACGCATCACTCTGAGCTGTAGGGCATCACAGTCTGTTACCACAGAT CTGGCTTGGTATCAACAAATGCCTGGGCAGGCCCCGCGACTGTTGATTTATGACGCC TCTACGCGGGCCACAGGATTTCCTGCCCGGTTCTCCGGGTCTGGTTCTGGCACCGAT TTTACCTTGACAATCAGTAGCTTGCAGGCAGAA GATTTCGCTGTGTATTACTGCCAA CATTATAAGACATGGCCTTTGACATTCGGCGGGGGAACCAAAGTGGAGATCAAACG CGCCGCAGCCCTGGACAATGAGAAGTCTAATGGGACCATCATTCACGTCAAAGGGA AACACCTGTGCCCCTCTCCTCTGTTCCCAGGCCCTTCTAAGCCCTTCTGGGTTCTCGT GGTGGTGGGCGGTGTCCTGGCCTGCTATTCCCTTCTTGTGACAGTG GCCTTTATCATT TTTTGGGTGAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACT CCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGA TTTCGCTGCCTATCGGAGCCGAGTGAAATTTTTCTAGATCAGCTGATGCTCCCGCCTA TCAGCAGGGACAGAATCAACTTTACAATGAGCTGAACCTGGGTCGCAGAGAAGGT ACGACG (SE Q Ш NO: 81)

Design 11F11 CD28T AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGAYYWT WIRQHPGKGLEWIGYIHYSGSTYSNPSLKSRITISLDTSKNQFSLKLNSVTAADTAVYYC ARQED YGGLFDYWGQGTLVTVS SGGGGSGGGGSGGGGSEIVMTQ SPATES VSPGERITL SCRASQSVTTDLAWYQQMP GQAPRLLIYDASTRATGFPARFSGSGSGTDFTLTISSLQAE

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DFAVYYCQHYKTWPLTFGGGTKVEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSK PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR (SEQ ID NO: 82)

11F11 CD8 DNA construct (signal sequence in bold)

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCC GCACGCCCGCAGGTACAGTTGCAGGAAAGCGGCCCCGGCCTTGTAAAACCAAGCC AGACTCTCAGTTTGACTTGCACCGTCTCAGGAGGAAGCATTTCCAGTGGGGCTTATT ATTGGACTTGGATTCGGCAGCATCCTGGGAAAGGGTTGGAATGGATCGGTTATATTC ATTATAGCGGTAGCAC CTATTCCAATCCGTCTTTGAAAAGCAGAATCACTATTTCAC TCGACACCTCTAAGAACCAGTTCAGTCTCAAACTGAACTCCGTGACAGCGGCCGAC ACAGCTGTGTACTACTGTGCACGGCAAGAAGATTATGGGGGGCTGTTCGATTATTGG GGCCAAGGCACACTGGTGACAGTATCAAGCGGTGGAGGAGCTCCGGGGGCGGAG GAAGTGGAGGCGGGGGGAGCGAAATTGTG ATGACCCAGTCTCCAGCCACGCTGTCA GTGTCTCCGGGAGAACGCATAACCCTCTCCTGCCGGGCCAGTCAGTCCGTCACGACC GATTTGGCTTGGTATCAACAGATGCCTGGGCAGGCCCCCCGCTTGCTGATCTATGAC GCCTCCACCAGCAACTGGTTTCCCCGCCCGGTTCAGCGGATCTGGAAGCGGTACA GATTTTACACTTACCATCTCATCATTGCAAGCTGAGGATTT TGCCGTGTACTACTGCC AGCACTACAAGACCTGGCCTTTGACGTTCGGCGGCGGAACAAAAGTGGAGATTAAA AGAGCCGCTGCCCTCAGTAACTCAATCATGTACTTTAGTCACTTTGTGCCTGTGTTTC TGCCAGCAAAGCCAACAACCACACCAGCACCCCGCCCTCCAACGCCTGCCCCAACC ATCGCCTCCCAGCCTCTGAGCTTGAGGCCTGAGGCTTGTCGCCCAGCTGCTGGAGGT GCTGTGCATACACGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACCACTT GCCGGTACTTGTGGTGTGTTGCTGCTCTCACTGGTCATCACGCTGTACTGTAACCATA GGAATAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCA CGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTC GCTGCCTATCGGAGCCGAG TGAAATTTTTCTAGATCAGCTGATGCTCCCGCCTATCAG CAGGGACAGAATCAACTTTACAATGAGCTGAACCTGGGTCGCAGAGAAGAGTACGA CGTTTTGGACAAACGCCGGGGCCGAGATCCTGAGATGGGGGGGAAGCCGAGAAGG AAGAATCCTCAAGAAGGCCTGTACAACGAGCTTCAAAAAGACAAAATGGCTGAGGC GTACTCTGAGATCGGCATGAAGGGCGAGCGGAGACGAGG CAAGGGTCACGATGGCT

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TGTATCAGGGCCTGAGTACAGCCACAAAGGACACCTATGACGCCCTCCACATGCAG GCACTGCCCCACGCTAG (SEQ ID NO: 83)

Design 11F11 CD8 AA (signal sequence in bold)

MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGAYYWT WIRQHPGKGLEWIGYIHYSGST YSNP SLKSRITISLDT SKNQF SLKLNS VTAADT AVYYC ARQEDYGGLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERITL SCRASQSVTTDLAWY QQMPGQAPRLLIYDASTRATGFPARFSGSGSGTDFTLTISSLQAE DFAVYYCQHYKTWPLTFGGGTKVEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRSRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 84)

Human FLT3 NM 004119 AA

MPALARDGGQLPLLVVFSAMIFGTITNQDLPVIKCVLINHKNNDSSVGKSS SYPMVSESPEDLGCALRPQSSGTVYEAAAVEVDVSASITLQVLVDAPGNISCLWVFKHS SLNCQPHFDLQNRGVVSMVILKMTETQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRP YFRKMENQDALVCISESVPEPIVEWVLC DSQGESCKEESPAVVKKEEKVLHELFGTDIRC CARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELENK ALEEGNYFEMSTYSTNRTMIRILF AF VS S VARNDTGYYTC S S SKHPSQS ALVTIVEKGF IN ATNS SEDYEIDQYEEFCF S VRFK AYPQIRCTWTF SRKS FPCEQKGLDNGYSISKFCNHKH QPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDK SPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILL NSPGPFPFIQDNISFYATIGVCLLFIVVLTLLICHKYKKQFRYESQLQMVQVT GSSDNEYF YVDFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKE KADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLLNYLRSKR EKFHRT WTEIFKEHNF SF YPTF Q SHPNS SMPGSRE VQIHPD SDQIS GLHGNSFHS EDEIES DGRVSECPHTYQNRRPFSREMDLGLLSPQAQVEDS (SEQ Ш NO: 85)

DNA with CAR signal peptide

ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCA CGCCCG (SEQ ID NO: 86)

CAR signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO: 87) scFv O45 linker DNA

GGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCC (SEQ ID NO: 88) scFv G4s linker: GGGGSGGGGSGGGGS (SEQ ID NO: 89)

scFv Whitlow DNA linker

GGGTCTACATCCGGCTCCGGGAAGCCCGGAAGTGGCGAAGGTAGTACAAAGGGG (SEQ ID NO: 90)

scFv Whitlow Linker: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 91)

4-IBB nucleic acid sequence (intracellular domain) AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAAC ACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ Ш NO: 92)

4-IBB AA (intracellular domain)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ Ш NO: 93)

0X40AA

RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 94)

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Incorporation by reference

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically and separately identified for inclusion by reference. However, the citation of a reference herein should not be construed as an endorsement that such reference is prior art to the present invention. To the extent that any of the definitions or terms presented in the references incorporated by reference differ from the terms and discussions presented herein, these rules and definitions shall control.

Equivalents

The above written description is considered sufficient to enable one skilled in the art to practice the present invention. The above description and examples specify certain preferred embodiments of the present invention and describe the best mode considered by the inventors of the present invention. However, it should be understood that, regardless of how detailed the above text is set forth, the present invention may be practiced in a variety of ways, and the present invention should be construed in accordance with the appended claims and any equivalents thereof.

The following examples, including experiments performed and results achieved, are presented for illustrative purposes only and are not intended to limit the present invention.

Example 1.

Namalwa, MV4;11 and HL60 cells (ATCC) and EoL1 cells (Sigma-Aldrich) were cultured in RPMI1640 (Lonza) + 10% FBS (Corning) + 1X penicillin streptomycin L-glutamine (Corning) (R10) and maintained at density cells from 0.5-2.0x106 cells/ml. To examine cell surface expression of FLT3, cells were incubated with anti-FLT3 antibody (BD Pharmingen) or anti-IgG1 isotype control antibody (BD Pharmingen) in stained buffer (BD Pharmingen) for 30 min at 4°C. Cells were then washed and resuspended in propidium iodide staining buffer (BD Pharmingen) before data acquisition. Expression of FLT3 on target cells is shown in FIG. 1.

Example 2.

Plasmids encoding the T7 promoter, CAR construct, and beta-globin stabilizing sequence were linearized by fermenting 10 μg of DNA with EcoRI and BamHI (NEB) overnight. The DNA was then fermented for 2 h at 50°C with proteinase K (Thermo Fisher, 600 U/ml), purified with phenol/chloroform, and precipitated by adding sodium acetate and two volumes of ethanol. The pellets were then dried, resuspended in RNase/DNase-free water and quantified using NanoDrop. One μg of linear DNA was then used for in vitro transcription using mMESSAGE mMACHINE T7 Ultra (Thermo Fisher) following the manufacturer's instructions. RNA was further purified using the MEGAClear kit (Thermo Fisher) following the manufacturer's instructions and counted using NanoDrop. mRNA integrity was assessed using agarose gel mobility. PBMCs were isolated from healthy donor bluish packs (Hemacare) using centrifugation to increase Ficoll pack density according to the manufacturer's instructions. PBMCs were stimulated using OKTZ (50 ng/ml, Miltenyi Biotec) in R10 + IL-2 medium (300 IU/ml, Proleukin®, Prometheus® Therapeutics and Diagnostics). 7 days after stimulation, T cells were washed twice in Opti-MEM medium (Thermo Fisher Scientific) and resuspended at a final concentration of 2.5 x 10 ⁷ cells/ml in Opti-MEM medium. 10 μg of mRNA was used for electroporation. Cell electroporation was performed using a Gemini X2 system (Harvard Apparatus BTX) to deliver a single pulse of 400 V for 0.5 ms in 2 mm cuvettes (Harvard Apparatus BTX). Cells were immediately transferred to R10 + IL-2 medium and allowed to recover for 6 h. To assess CAR expression, T cells were stained with FLT-=3-HIS (Sino Biological Inc.) or biotinylated protein L (Thermo Scientific) in stained buffer (BD Pharmingen) for 30 min at 4°C. Cells were then washed and stained with anti-HIS-PE (Miltenyi Biotec) or PE Streptavidin (BD Pharmingen) in staining buffer for 30 min at 4°C. Cells were then washed and resuspended in stained buffer using propidium iodide (BD Pharmingen) before data acquisition. Expression of FLT3 CAR in electroporated T cells is shown in FIG. 2.

Example 3.

To assess cytolytic activity in electroporated FLT3 CAR T cells, effector cells were cultured with target cells at an E:T ratio of 1:1 in R10 medium. After 16 h of coculture, supernatants were analyzed by Luminex (EMD Millipore) and target cell viability was assessed by flow cytometric analysis of propidium iodide (PI) uptake by CD3-negative cells. The cytolytic activity of electroporated CAR T cells is shown in FIG. 3, and the production of cytokines is shown in FIG. 4.

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Claims (18)

Example 4. A third generation lentiviral transfer vector containing various CAR constructs was used with ViraPower Lentiviral Packaging Mix (Life Technologies) to generate lentiviral supernatants. Briefly, the transform mixture was formed by mixing 15 μg of DNA and 22.5 μl of polyethylenimine (Polysciences, 1 mg/ml) in 600 μl of OptiMEM medium. The mixture was incubated for 5 min at room temperature. Simultaneously, 293 T cells (ATCC) were trypsinized, counted, and a total of 10 x 106 cells were seeded into a T75 vessel along with the transform mixture. 3 days after transformation, supernatants were collected and filtered through a 0.45 μm filter and stored at −80°C until use. PBMCs were isolated from healthy donor bluish packs (Hemacare) using centrifugation to increase Ficoll pack density according to the manufacturer's instructions. PBMCs were stimulated with OKT3 (50 ng/ml, Miltenyi Biotec) in R10 + IL-2 medium (300 IU/ml, Proleukin®, Prometheus® Therapeutics and Diagnostics). 48 h after stimulation, cells were transfected using lentivirus at an MOI of 10. Cells were maintained at 0.5-2.0 x 106 cells/ml before using activity assays. To assess CAR expression, T cells were stained with FLT-3-HIS (Sino Biological Inc.) or biotinylated protein L (Thermo Scientific) in staining buffer (BD Pharmingen) for 30 min at 4°C. Cells were then washed and stained with anti-HIS-PE (Miltenyi Biotec) or PE Streptavidin (BD Pharmingen) in staining buffer for 30 min at 4°C. Cells were then washed and resuspended in stained buffer using propidium iodide (BD Pharmingen) before data acquisition. FLT3 CAR expression in T cells from two healthy donors is shown in FIG. 5. Example 5. To assess cytolytic activity in FLT3 CAR T cells transfected with lentivirus, effector cells were cultured with target cells at an E:T ratio of 1:1 in R10 medium. After 16 h of coculture, supernatants were analyzed by Luminex (EMD Millipore) and target cell viability was assessed by flow cytometric analysis of propidium iodide (PI) uptake by CD3-negative cells. The average cytolytic activity of lentivirus-transfected CAR T cells from two healthy donors is shown in Fig. 6, and cytokine production by CAR T cells from each healthy donor is shown in FIG. 7. Example 6. To assess CAR T cell proliferation in response to FLT3-expressing target cells, T cells were marked by CFSE before coculture with target cells at a 1:1 E:T ratio in R10 medium. After 5 days, T cell proliferation was assessed by flow cytometric CFSE dilution analysis. Proliferation of FLT3 CAR T cells is shown in FIG. 8. Example 7. To evaluate in vivo anti-leukemia activity, FLT3 CAR T cells were generated for use in a xenogeneic human AML model. CAR expression of various effector lines used in a xenogeneic human AML model is shown in FIG. 9. Luciferase-labeled MV4;11 cells (2x10 ⁶ /animal) were intravenously injected into 5-6 week old female NSG mice. After 6 days, 6x106 T cells (~50% CAR+) in 200 μl PBS were injected intravenously and the tumor burden of the animals was measured weekly using bioluminescence imaging. As shown in FIG. 10, injection of 10E3-CD28T and 8B5-CD28T CAR-expressing CAR T cells significantly reduced tumor burden at all time points assessed. As shown in FIG. 11, this was further supported by a survival assay where injection of 10E3-CD28T or 8B5-CD28T CAR-expressing CAR T cells provided a significant survival benefit among animals that received Mock-transformed cells or CAR T cells expressing the 10E3-CD28 or 10E3- CD8. No significant differences were observed between the 10E3-CD28T and 8B5-CD28T designs in terms of efficacy. CLAIM 1. A chimeric antigen receptor that specifically binds to FLT3, wherein the chimeric antigen receptor includes an antigen binding molecule that specifically binds to FLT3, a costimulatory domain, and an activating domain that is a CD3 zeta signaling domain, wherein the antigen binding molecule comprises a heavy chain variable region containing complementarity determining regions (CDRs) 1, 2 and 3 having the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and a light chain variable region containing CDRs 1, 2 and 3 having the amino acid the sequences are SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or the VH region of SEQ ID NO: 16 and the VL region of SEQ ID NO: 21; - 52 044866 where the VH and VL regions are linked by at least one linker, where the linker is a scFv G4S linker or a scFv Whitlow linker. 2. The chimeric antigen receptor according to claim 1, wherein the costimulatory domain is a signaling region CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD1), inducible costimulator T -cells (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C , Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, receptor for Toll ligands, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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, or a ligand that specifically binds CD83. 3. The chimeric antigen receptor of claim 2, wherein the CD28 co-stimulatory domain comprises a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue from the sequence in SEQ ID NO : 2, SEQ ID NO: 4 or SEQ ID NO: 4 in combination with SEQ ID NO: 6 or SEQ ID NO: 8, or the CD8 costimulatory domain includes a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue from the sequence in SEQ ID NO: 14. 4. The chimeric antigen receptor of claim 2, wherein CD3 zeta comprises a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue from the sequence in SEQ ID NO: 10. 5. The chimeric antigen receptor according to any one of claims 1 to 4, wherein the co-stimulatory domain includes a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue from the sequence in SEQ ID NO: 2, and the activating domain includes a sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue from the sequence in SEQ ID NO: 10. 6. A polynucleotide encoding a chimeric antigen receptor according to any one of claims 1 to 5. 7. An expression vector comprising the polynucleotide of claim 6, wherein the expression vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-linked vector, or a lentiviral vector. 8. An immune cell expressing a chimeric antigen receptor that specifically binds to FLT3, comprising the expression vector of claim 7. 9. The immune cell of claim 8, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR expressing cell, a dendritic cell, or an NK T cell. 10. The immune cell of claim 9, wherein the cell is an autologous T cell. 11. The immune cell of claim 9, wherein the cell is an allogeneic T cell. 12. The immune cell according to claim 8, wherein the expression vector is introduced into a cell that is isolated from the patient's body or the donor's body or that is grown from a sample taken from the patient's body or the donor's body. 13. Pharmaceutical composition containing an immune cell according to any one of claims 8-12. 14. A method of treating a FLT3-related disease or disorder in a subject in need thereof, comprising administering to the subject a chimeric antigen receptor according to any one of claims 1 to 5, or administering to the subject a cell according to any one of claims 8 to 12, or administering to the subject a pharmaceutical composition according to paragraph 13. 15. The method of claim 14, wherein the disease or disorder is cancer. 16. The method of claim 15, wherein the cancer is leukemia, lymphoma or myeloma. 17. The method of claim 14, wherein the disease or disorder is at least one of the following: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia , acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorder, myeloid neoplasm, myeloid sarcoma and inflammatory/autoimmune disease. 18. The method of claim 17, wherein the inflammatory/autoimmune disease is at least one of the following: rheumatoid arthritis, psoriasis, allergies, asthma, Crohn's disease, IBD (inflammatory bowel disease), IBS (irritable bowel syndrome), fibromyalgia , mastocytosis and celiac enteropathy. -