Bispecific polypeptides for engagement of car expressing immune cells with antigen presenting cells and uses thereof

Abstract

To provide novel reagents to improve the in vitro and in vivo expansion of CAR T cells for ACT.SOLUTION: The present application is directed to bispecific polypeptides comprising a first binding domain binding an antigen on an antigen presenting cell (ARC) and a second binding domain binding an antigen on an immune cell expressing a chimeric antigen receptor (CAR). Nucleic acids, vectors and host cells used in producing the polypeptide of the invention are also disclosed. Compositions comprising the bispecific polypeptides and methods of treating cancer and stimulating activation and expansion of immune cells in vivo and in vitro are also disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates generally to bispecific polypeptides, including portions thereof, that comprise a first binding domain capable of binding to an antigen-presenting cell (APC) and a second binding domain capable of binding to a T cell, and uses thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Australian Provisional Patent Application Nos. 2018904117, filed October 30, 2018, and 2019903255, filed September 4, 2019, the entire contents of which are hereby incorporated by reference herein. [Background technology]

[0003] Adoptive cell transfer (ACT) is demonstrating exciting potential for cancer treatment. In ACT, large numbers of autologous tumor-reactive T cells are generated in vitro before being reinfused into the patient. Tumor-reactive T cells can be isolated from blood or tumors and expanded in vitro using peptide and / or cytokine stimulation. The most impressive responses to ACT to date have been observed in specific tumor types, such as melanoma, where T cell transfer, along with a preconditioning regimen including IL-2 administration and chemotherapy and / or total body irradiation, produced objective responses in 52 of 93 patients (56%); 20 of the 93 patients achieved complete remission, with 19 of those 20 patients showing ongoing durable complete remissions more than 5 years after treatment (Non-Patent Document 1). Patients with Epstein-Barr virus (EBV)-associated lymphoproliferative disease after bone marrow transplantation can also benefit from ACT, with virtually all patients achieving complete disease regression after adoptive transfer of EBV-specific T cells (Non-Patent Document 2). However, the isolation of autologous T cells with responsiveness against other types of cancer is rare.

[0004] Methods for improving T cell responsiveness to ACT include genetic modification of patient lymphocytes to generate tumor-responsive T cells for most malignancies, including solid and hematological cancers. Two major approaches to genetic modification involve genes encoding T cell receptors (TCRs) or chimeric antigen receptors (CARs). CARs consist of antibody-derived domains fused with T cell signaling domains that redirect T cell effector function toward tumor cells. While both approaches can render T cells tumor-responsive, the non-MHC-restricted CAR approach may be more widely applicable to a wider range of patients.

[0005] CARs can take various forms (Non-Patent Document 3), but typically consist of an extracellular domain consisting of a single-chain variable fragment (scFv) of an antibody specific for a tumor-associated antigen (TAA). This scFv is linked via a hinge domain and a transmembrane domain to an intracellular region consisting of one or more signaling moieties. CARs have been developed with specificity against a range of TAAs, including Her2, CEA, FBP, CD19, and CD209. The most advanced clinical trials have utilized CARs specific for CD19 to treat B-cell leukemia and lymphoma (Non-Patent Document 3). In 2017, two of these CD19-CAR T-cell therapies were approved by the U.S. FDA for the treatment of B-cell malignancies (Non-Patent Document 4). Despite these results, few objective responses have been reported for solid tumors, which may be due to insufficient activation, proliferation, and persistence of CAR T cells and / or an immunosuppressive tumor microenvironment.

[0006] Attempts to optimize this type of therapy have led to the combination of CAR T cells with other therapeutic approaches designed to overcome tumor-induced immunosuppression, including co-treatment with α-PD-1 monoclonal antibodies (NPL 5), genetic modification of signaling and cytokine pathways (NPL 6), and the use of adjuvants such as agonistic α-4-1BB monoclonal antibodies (mABs) (NPL 7), bispecific T cell engagers (BiTEs), etc. Some of these approaches have proven successful in enabling direct interactions between T cells and cancer cells, which can lead to T cell proliferation in hematopoietic cancer cells, although significant proliferation has rarely been observed in the setting of solid tumors (NPL 8). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hinrichs et al.,2014,Immunology Reviews,257(1):56-71 [Non-patent document 2] Heslop et al.,2010,Blood,115(5):925-35 [Non-patent document 3] Kershaw et al.,2013,Nature Reviews Cancer,13(8):525-41 [Non-patent document 4] Kymriah, Novartis and Yescarta, Kite Pharma / Gilead [Non-patent document 5] John et al.,2013,Clinical Cancer Research,19:5636-46 [Non-patent document 6] Koneru et al.,2015,Oncoimmunology,4:e994446 [Non-Patent Document 7] Mardiana et al.,2017,Cancer Research [Non-patent document 8] Huehls et al.,2015,Immunological Cell Biology,93(3):290-6 Summary of the Invention [Problem to be solved by the invention]

[0008] Optimizing therapeutic approaches for delivering CAR T cells is often adversely affected by the difficulties encountered in expanding and generating CAR T cells in vitro due to the low lymphocyte counts and poor condition of cells from patients with a history of multiple drug therapies (U.S. Food and Drug Administration: KYMRIAH (Tisagenlecleucel), August 30, 2017). Furthermore, even when sufficient donor cells are available, significant numbers of cells must be generated to provide patients with effective doses of these therapies; allogeneic cells from healthy donors have been suggested as a solution to this problem, but they present numerous challenges, such as human leukocyte antigen (HLA) mismatches between donor and recipient, which can lead to graft-versus-host disease (GvHD). Thus, there remains a pressing need for the development of novel reagents to improve the in vitro and in vivo expansion of CAR T cells for ACT. [Means for solving the problem]

[0009] In one aspect disclosed herein, there is provided a bispecific polypeptide comprising a first binding domain and a second binding domain, wherein the first binding domain is an antibody or antibody fragment that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and the second binding domain is an antibody or antibody fragment that specifically binds to an antigen on an immune cell expressing a chimeric antigen receptor (CAR).

[0010] In one aspect disclosed herein, there is provided a bispecific polypeptide comprising a first binding domain and a second binding domain, wherein the first binding domain is an antibody or antibody fragment that specifically binds to an antigen expressed on an antigen-presenting cell (APC), and the second binding domain is an antibody or antibody fragment that specifically binds to a chimeric antigen receptor (CAR) expressed by an immune cell.

[0011] In another aspect of the present disclosure, there is provided a nucleic acid encoding a bispecific polypeptide as described herein.

[0012] In another aspect of the present disclosure, there is provided a vector comprising a nucleic acid sequence described herein operably linked to a control sequence.

[0013] In another aspect of the present disclosure, there is provided a cell comprising the vector described herein.

[0014] In another aspect disclosed herein, there is provided a method for producing a bispecific polypeptide, the method comprising: (i) culturing a cell as described herein in a culture medium and under conditions suitable for expression of the bispecific polypeptide; and (ii) isolating the expressed bispecific polypeptide from the cell or culture medium.

[0015] In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising a bispecific polypeptide as described herein and a pharmaceutically acceptable carrier.

[0016] In another aspect disclosed herein, there is provided a method for treating cancer, comprising co-administering to a subject in need thereof therapeutically effective amounts of: (i) immune cells expressing a CAR; and (ii) a bispecific polypeptide or pharmaceutical composition disclosed herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the subject's endogenous APCs and an antigen on the immune cells, preferably the antigen on the immune cells is the antigen on the CAR, thereby stimulating the in vivo activation and proliferation of the immune cells to treat the cancer.

[0017] In another aspect disclosed herein, there is provided a method for stimulating immune cell activation and proliferation in vivo, comprising co-administering to a subject effective amounts of: (i) immune cells expressing a CAR; and (ii) a bispecific polypeptide or pharmaceutical composition disclosed herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the subject's endogenous APC and an antigen on the immune cell, preferably the antigen on the immune cell is the antigen on the CAR, thereby stimulating immune cell activation and proliferation in vivo.

[0018] In another aspect disclosed herein, there is provided a method for stimulating immune cell activation and proliferation in vitro, comprising: culturing isolated immune cells expressing a CAR in a culture medium comprising: (i) an APC or an APC mimetic, and (ii) a bispecific polypeptide disclosed herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the APC or APC mimetic and an antigen on the immune cell, preferably the antigen on the immune cell is the antigen on the CAR, thereby stimulating immune cell activation and proliferation in vitro.

[0019] In another aspect of the present disclosure, there is provided the use of a bispecific polypeptide or pharmaceutical composition as described herein in the manufacture of a medicament for treating cancer.

[0020] In yet another aspect, there is provided a bispecific polypeptide or a pharmaceutical composition as described herein for use in the treatment of cancer.

[0021] In yet another aspect, the present invention provides kits or articles of manufacture comprising one or more bispecific polypeptides of the invention, nucleic acids encoding said bispecific polypeptides and / or pharmaceutical compositions as described above.

[0022] In another aspect, there is provided a kit for use in the therapeutic applications described above, comprising: (a) a container holding a bispecific polypeptide, nucleic acid, vector, or pharmaceutical composition of the invention; and (b) A label or package insert containing instructions for use. A kit is provided comprising:

[0023] In certain embodiments, the kit may further comprise one or more active ingredients or components for treating cancer, for example, the kit may further comprise immune cells expressing a CAR. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows that binding domains specific for antigens on CAR T cells can be expressed using the Expi293 cell expression system. [Figure 2] Figure 1 shows that full-length bispecific polypeptides can be expressed using the 293T cell line and the Lipofectamine system. (A-B) Photographic images of Western blots for the His tag of protein lysates from 293T cells expressing BEAT1 (9E10-M5 / 114). [Figure 3] Figure 1 shows that full-length bispecific polypeptides can be expressed using the 293T cell line and the jetPEI system. (A-B) Photographic images of Western blots for the His tag of protein lysates from 293T cells expressing BEAT2 (MHCII / c-myc) and BEAT3 (CD40 / CD3). [Figure 4]Figure 1 shows that full-length bispecific polypeptides can be expressed and released from cells after cell homogenization. (A) Photographic images of Western blots for His-tags of protein lysates from 293T cells expressing BEAT2 (MHCII / c-myc) and BEAT3 (CD40 / CD3) from homogenized 0.75% Super+ cells from a 6-well plate and (B) non-homogenized cell pellet from a 6-well plate. [Figure 5] Figure 1 shows that the bispecific polypeptide Staphylococcus Enterotoxin B (SEB) mediates T cell proliferation and IFN-γ secretion. (A) Graphical representation of T cell proliferation (y-axis) in splenocytes from B6 mice or human PBMCs in the presence or absence of SEB (x-axis). (B) Graphical representation of IFN-γ secretion (y-axis) in splenocytes from B6 mice or human PBMCs in the presence or absence of SEB (x-axis). (C) Graphical representation of the percentage of TCR-Vβ8+ cells (x-axis) in the spleens of mice bearing established E0771-Her2 mammary tumors after 48 hours in the presence or absence of SEB. *p<0.05, ***p<0.001 (Student's t-test). [Figure 6A] Figure 1 shows that the anti-tumor efficacy of CAR T cells is enhanced by co-administration of SEB in vivo. (A) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer. (B) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established 24JK-Her2 breast cancer. (C) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established MC38-Her2 breast cancer. Error bars: SEM. *p<0.05, **p<0.01 (Student's t-test). (D) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer, including mice receiving just SEB alone or CAR T cells alone. Unless otherwise stated, significance was compared between the untreated and SEB+CAR groups. [Figure 6B] Figure 1 shows that the anti-tumor efficacy of CAR T cells is enhanced by co-administration of SEB in vivo. (A) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer. (B) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established 24JK-Her2 breast cancer. (C) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established MC38-Her2 breast cancer. Error bars: SEM. *p<0.05, **p<0.01 (Student's t-test). (D) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer, including mice receiving just SEB alone or CAR T cells alone. Unless otherwise stated, significance was compared between the untreated and SEB+CAR groups. [Figure 6C] Figure 1 shows that the anti-tumor efficacy of CAR T cells is enhanced by co-administration of SEB in vivo. (A) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer. (B) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established 24JK-Her2 breast cancer. (C) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established MC38-Her2 breast cancer. Error bars: SEM. *p<0.05, **p<0.01 (Student's t-test). (D) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer, including mice receiving just SEB alone or CAR T cells alone. Unless otherwise stated, significance was compared between the untreated and SEB+CAR groups. [Figure 6D]Figure 1 shows that the anti-tumor efficacy of CAR T cells is enhanced by co-administration of SEB in vivo. (A) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer. (B) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established 24JK-Her2 breast cancer. (C) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established MC38-Her2 breast cancer. Error bars: SEM. *p<0.05, **p<0.01 (Student's t-test). (D) Graphical representation of tumor size (mm2; y-axis) versus time (days after treatment; x-axis) in mice bearing established E0771-Her2 breast cancer, including mice receiving just SEB alone or CAR T cells alone. Unless otherwise stated, significance was compared between the untreated and SEB+CAR groups. [Figure 7] Figure 1 shows that co-administration of SEB enhances CAR T cell infiltration into tumors. Graphical representation of cells stained with CD8 in the absence (A) or presence (B) of SEB. In CAR+SEB, dark staining represents infiltrating CAR T cells (i.e., CD8+). Scale bar: 50 μm. [Figure 8] Figure 1 shows that binding of CAR-specific binding domains does not interfere with CAR-mediated T cell function. (A) Graphical representation of IFN-γ secretion (pg / mL; y-axis) of CAR T cells incubated with E0771-Her2 cells and control or anti-myc antibody (y-axis). (B) Graphical representation of cytotoxicity (% killing; y-axis) of CAR T cells co-cultured with E0771-Her2 cells in the presence or absence of anti-myc antibody (x-axis). [Figure 9] Figure 1 shows that bispecific polypeptides (i.e., BEAT) promote CAR T cell proliferation in vitro. Graphical representation showing cell counts of splenocytes from transgenic CAR mice labeled with CSFE and incubated with BEAT (anti-CD40 / anti-myc) or a mixture of anti-CD40 and anti-myc antibodies for 72 hours. [Figure 10]Figure 1 shows that BEAT mediates CAR T cell activation and proliferation. Three examples of BEAT were generated by chemically conjugating an antibody specific for the myc tag (present in the CAR) to an antibody specific for either CD40, PD-L2, or Clec9a expressed on various subsets of APCs. Mouse CAR T cells were incubated with mouse splenocytes (as the source of APCs) in the presence or absence of BEAT. T cells lacking CAR expression were used as a control. Incubation of CAR T cells with APCs in the presence of unconjugated antibodies (listed) was also used as a control. (A) In the presence of each BEAT, CAR T cells (right panel) secrete IFN-γ, whereas control T cells (left panel) do not. (B) CAR T cells were loaded with the fluorescent dye CFSE and incubated with APCs in the presence or absence of each alternative BEAT. The extent of proliferation is evident from the decrease in fluorescence intensity of the transfected CAR T cells (right panel) compared to control non-transfected T cells (left panel). [Figure 11] Figure 1 shows that CD40-myc BEAT enables eradication of E0771-Her2 breast cancer in mice. Her2-transgenic mice bearing established subcutaneous E0771-Her2 tumors were treated with CAR T cells (2x106, iv) and / or BEAT specific for myc-CD40 (36 μg, ip on day 0). Other groups of mice received unconjugated anti-myc and anti-CD40 antibodies or were left untreated (control). (A) Tumor growth rate and (B) mouse survival rate. Data represent three independent experiments with six mice per group. *p<0.05, ***p<0.001, unpaired Student's t-test. [Figure 12A]Figure 1 shows that CD40-myc BEAT enables the accumulation of T cells within tumors. Mice bearing subcutaneous E0771-Her2 tumors were administered anti-HER2 CAR T cells (2 × 10 , iv) together with CD40-myc BEAT (36 μg, ip on day 0). Other mice received CAR T cells or BEAT alone, or were left untreated (control). (A) Seven days after the start of treatment, tumors were harvested, dissociated, stained to detect CD8+ T cells, and analyzed using flow cytometry. Data represent three individual mice per group. (B) Tumors were formalin-fixed, sectioned, and stained with DAPI (blue) and anti-CD8 (brown). Images are shown representing three sections from mice receiving the listed treatments. [Figure 12B] Figure 1 shows that CD40-myc BEAT enables the accumulation of T cells within tumors. Mice bearing subcutaneous E0771-Her2 tumors were administered anti-HER2 CAR T cells (2 × 10 , iv) together with CD40-myc BEAT (36 μg, ip on day 0). Other mice received CAR T cells or BEAT alone, or were left untreated (control). (A) Seven days after the start of treatment, tumors were harvested, dissociated, stained to detect CD8+ T cells, and analyzed using flow cytometry. Data represent three individual mice per group. (B) Tumors were formalin-fixed, sectioned, and stained with DAPI (blue) and anti-CD8 (brown). Images are shown representing three sections from mice receiving the listed treatments. [Figure 13] Figure 1 shows that CAR T cells persist long-term in mice. Spleens were harvested from mice surviving long-term (>200 days) after eradication of E0771-Her2 tumors by CAR T cells + CD40-myc BEAT (left panel) or from naive mice (right panel) and stained with anti-myc and CD8 to detect CAR T cells. Data are representative of 3 mice. [Figure 14]This figure shows that mice that had rejected Her2-positive tumors after CAR T cell + BEAT therapy were completely resistant to rechallenge with Her2-positive tumors. Long-term survivors who eradicated subcutaneous E0771-Her2 tumors mediated by Her2-specific CAR T cells and CD40-myc BEAT were rechallenged with the same Her2-positive tumor cells (5 × 10 ) by subcutaneous injection on the opposite flank. Tumor growth rate (upper panel) and mouse survival rate (lower panel). Tumor growth was visualized in naive mice as a control. (8 mice in the rechallenge group and 6 mice in the control group; ***p<0.001, ****p<0.0001). [Figure 15] Her2-negative tumor growth is inhibited in mice that had previously rejected Her2-positive tumors mediated by CAR T cells and BEAT therapy. Mice that had previously rejected E0771-Her2 tumors after treatment with Her2-specific CAR T cells and CD40-myc BEAT were re-challenged on the contralateral flank with identical E0771 cells (5 x 10 cells) lacking Her2 expression. Tumor growth in naive mice is shown as a control. Tumor growth rate (upper panel) and mouse survival rate (lower panel) are shown. (15 mice in the re-challenged group vs. 5 mice in the control group; **p<0.01, ***p<0.001, ****p<0.0001). [Figure 16] Figure 1 shows that anti-Her2 CAR T cells in combination with CD40-myc-conjugated BEAT inhibit the growth of MC38-Her2 colon carcinoma in mice. Mice with established subcutaneous MC38-Her2 tumors were treated with anti-Her2 CAR T cells (2x106, iv) and CD40-myc BEAT (36 μg on day 0). Other groups of mice received CAR T cells or the (listed) antibody alone, or were left untreated (control). There were 6 mice per group, except for the BEAT+CAR group, which had 7 mice. **p<0.01). [Figure 17]Figure 1 shows that PD-L2-myc and Clec9a BEAT conjugates enable CAR T cell-mediated inhibition of tumor growth. BEATs specific for the APC-expressed molecules PD-L2 or Clec9a were prepared by chemical conjugation of monoclonal antibodies to anti-myc antibodies. Mice bearing established subcutaneous E0771-Her2 tumors received the listed treatments or were left untreated (control) (3-6 mice per group; *p<0.05, **p<0.01). [Figure 18] Figure 1 shows that recombinant human CD40-myc BEAT mediates the proliferation of human CAR T cells. CAR T cells were generated from human peripheral blood using retroviral transduction of a Her2-specific CAR. CAR T cells (2 x 105 / ml) were labeled with CFSE and incubated for 5 days with or without chemically conjugated or recombinant CD40-myc BEAT (conjugated 3 μg / ml, recombinant 0.5 μg / ml) in the presence of 5 Gy-irradiated CTV-labeled human blood leukocytes as a source of APCs (left panel). T cells transfected with an empty vector served as a control (right panel). Cells were analyzed using flow cytometry. [Figure 19] Figure 1 shows that recombinant human CD40-myc BEAT enhances IFN-γ secretion from CAR T cells. Human CAR T cells were generated from peripheral blood using a retroviral vector encoding an anti-Her2 CAR. CAR T cells were incubated with human blood leukocytes as a source of APCs for 5 days in the presence or absence of chemically or recombinantly prepared CD40-myc BEAT. Anti-CD3 was included in some culture wells as a positive control. Supernatants were collected and analyzed for IFN-γ using ELISA. [Figure 20]Human CAR T cells combined with conjugated human BEAT inhibited tumor growth in mice. Nod-SCID-γ (NSG) mice bearing established subcutaneous MDA-MB-231 human mammary tumors received CAR T cells with or without conjugated human CD40-myc BEAT (day 0, 36 μg, i.p.). Human blood-derived leukocytes (1 × 10 ) were also delivered intravenously to serve as a source of APCs. One group of mice remained untreated as a control. (5 mice per group; *p<0.05, **p<0.01, ***p<0.001, Student's t-test). [Figure 21] BEAT can induce responses from T cells expressing CARs specific for a range of tumor antigens and CARs containing various antigens arranged at various positions within the CAR. T cells bearing a CAR for binding the tumor antigen CEA and a myc tag (CEA-myc CAR) secreted more IFN-γ (pg / mL; y-axis) when incubated with mouse splenocytes and a BEAT with a first binding domain for binding to myc and a second binding domain for binding to CD40. T cells bearing a CAR for binding the tumor antigen Her2 and a α-FLAG tag located at the N-terminus of the CAR (Her2-FLAG CAR) secreted more IFN-γ when incubated with mouse splenocytes and a BEAT with a first binding domain for binding to FLAG and a second binding domain for binding to CD40. BEAT specific for FLAG and CD40 also induced IFN-γ secretion from Her2-FLAG CAR T cells in the presence of CD40-expressing splenocytes. DETAILED DESCRIPTION OF THE INVENTION

[0025] Throughout this specification, unless the context requires otherwise, the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0026] The mention in this specification of any prior publication (or information derived therefrom) or of any publicly known material is not, and should not be taken as, any acknowledgement, admission or suggestion that the prior publication (or information derived therefrom) or publicly known material forms part of the general knowledge in the field of endeavor to which this specification pertains.

[0027] Unless otherwise defined, all technical and scientific terms used herein should be assumed to have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] Unless otherwise indicated, the molecular biology, cell culture, and laboratory techniques utilized herein are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M.G. Lover and B.D.H.Means (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F.M.A.usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub., all of which are incorporated herein by reference. The methods described herein are believed to be well known in the art and are provided for the convenience of the reader. All other publications mentioned herein are incorporated by reference in their entirety.

[0029] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" includes a single polypeptide as well as two or more polypeptides; reference to an "antigen-presenting cell (APC)" includes a single APC as well as two or more APCs, etc.

[0030] Amino acid and nucleotide sequences are referred to by sequence identification numbers (SEQ ID NOs), as shown in Table 1 below.

[0031] [Table 1]

[0032] The present disclosure is anticipated, at least in part, by the inventors' unexpected discovery that engagement of CAR T cells with antigen-presenting cells (APCs) using bispecific polypeptides enhances T cell proliferation in vitro and in vivo with non-toxic and high tumor inhibition, while reducing the effective dose of CAR T cells required to achieve a therapeutic effect. Importantly, CAR T cells receive activation and proliferation signals in lymphoid tissues, away from the immunosuppressive tumor microenvironment.

[0033] Thus, in one aspect disclosed herein, there is provided a bispecific polypeptide comprising a first binding domain and a second binding domain, wherein the first binding domain is an antibody or antibody fragment that specifically binds to an antigen expressed on an antigen APC, and the second binding domain is an antibody or antibody fragment that specifically binds to an antigen on a chimeric antigen receptor (CAR) expressed by an immune cell.

[0034] Bispecific Polypeptides As used herein, the term "bispecific polypeptide" refers to a polypeptide that can simultaneously specifically bind to two different target antigens. The bispecific polypeptides described herein contain two structurally distinct binding domains (i.e., regions), each of which specifically binds to a single target antigen. Bispecific polypeptides can be used to bind to a target antigen on an APC and a different target antigen on an immune cell expressing a CAR. Furthermore, bispecific polypeptides can be used to bind to a target antigen on an APC and a different target antigen on a CAR expressed by an immune cell. Bispecific polypeptides can comprise the polypeptide sequences (i.e., domains) of one or more antibodies or antibody fragments (e.g., one or more scFvs). In another embodiment, bispecific polypeptides can comprise the polypeptide sequences of one or more ligands.

[0035] In one embodiment, the bispecific polypeptide is a tandem single-chain variable fragment antibody (taFv) comprising a first scFv and a second scFv.

[0036] The bispecific polypeptides described herein can be variously referred to as "APC and T cell bispecific engagers" or "BEATs."

[0037] "Polypeptide," "peptide," "protein," and "proteinaceous molecule" are used interchangeably herein to refer to molecules comprising or consisting of polymers of amino acid residues and variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic, non-naturally occurring amino acid, e.g., a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0038] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, e.g., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones. An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid.

[0039] Amino acids may be referred to herein by their commonly used full names (e.g., cysteine), by their commonly known three letter symbols (e.g., Cys), or by the one-letter symbol recommended by the IUPAC-IUB biochemical nomenclature system (e.g., C). Nucleotides may likewise be referred to by their commonly accepted one-letter codes.

[0040] The term "antigen," as used herein, refers to a molecule bound by an "antibody," "antibody fragment," or "bispecific polypeptide" herein. An antigen can be a protein recognized by an immunoglobulin, in which case the site on the protein bound by the immunoglobulin is called an "epitope." In one embodiment, the antigen can be a ligand for a cell surface receptor (e.g., MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, and CD83). In another embodiment, the antigen is a tag on a CAR expressed by an immune cell.

[0041] As used herein, the term "antigen-presenting cell" or "APC" can be a professional antigen-presenting cell (e.g., a dendritic cell, a macrophage, a B cell, an epithelial cell, etc.) or a non-professional antigen-presenting cell (e.g., a fibroblast, a thymic epithelial cell, a thyroid epithelial cell, a glial cell, a pancreatic β cell, etc., a vascular endothelial cell, etc.). In a preferred embodiment, the APC is a professional antigen-presenting cell and is not a tumor cell.

[0042] In one embodiment, the first binding domain specifically binds to an antigen expressed on an APC selected from the group consisting of MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, and CD83. MHCII is expressed on dendritic cells (DCs), mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells; Clec9a is expressed on BDCA+ dendritic cells and a small subset of CD14+ / CD16- monocytes; PD-L1 and PD-L2 are expressed on macrophages, myeloid DCs, B cells, and vascular endothelial cells; galectin is expressed by T helper cells and B cells; CD11c is expressed at high levels by DCs; CD19 is expressed on all professional B cells to mature B cells and follicular DCs; CD40 is expressed by dendritic cells, B cells, and macrophages; and CD83 is expressed primarily by mature DCs. In a preferred embodiment, the antigen specifically bound by the first binding domain is not a tumor-associated antigen.

[0043] In one embodiment, the second binding domain specifically binds to an antigen on an immune cell expressing a CAR or similar receptor. In yet another embodiment, the second binding domain of the bispecific polypeptide of the invention is capable of binding to an antigen on an immune cell, where the antigen is not part of a CAR but is present on the cell surface of the immune cell.

[0044] In certain embodiments, the immune cells are typically genetically modified T cells or NK cells, where the antigen on the genetically modified T cells or NK cells is part of a CAR. According to these embodiments, the second binding domain of the bispecific polypeptide of the invention can bind to the extracellular portion of the CAR. The second binding domain can bind to the antigen-binding domain of the CAR, or the second binding domain can bind to an extracellular region of the CAR that is not involved in antigen binding.

[0045] In certain embodiments, the CAR present on the immune cell may further comprise additional amino acids or molecules for binding to the second binding domain. As is known in the art, CAR constructs can be designed to include a "tag," which is typically a short amino acid sequence that is specifically recognized by an antibody. In some embodiments, the immune cell is a T cell or NK cell genetically engineered to express a CAR that includes a tag. In the context of such embodiments, the second binding domain of the bispecific polypeptide may bind to the tag or may bind to a region of the CAR other than the tag. In some embodiments where the immune cell is a T cell or NK cell genetically engineered to express a CAR that includes a tag, the second binding domain of the bispecific polypeptide binds to a region of the CAR other than the tag.

[0046] Specific examples of tags present on the CAR include peptide tags (e.g., FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, Strep tag, eXact tag) and protein tags (e.g., GST tag, MBP tag, GFP tag, tags in the form of domains for binding to antibodies or other proteins, nucleoproteins (such as leucine zippers) or any other protein modification to the CAR). In one embodiment, the antigen or affinity tag on the CAR is a c-Myc tag.

[0047] In some embodiments, the immune cells are T cells or NK cells genetically engineered to express a tag-free CAR, hi some embodiments, the immune cells are T cells or NK cells genetically engineered to express a tag-free CAR, or any heterologous tumor-associated antigen or fragment of a tumor-associated antigen.

[0048] In some embodiments, the immune cell is a T cell or NK cell genetically engineered to express a CAR, and the bispecific polypeptide binds to the extracellular portion of the CAR. As is known in the art, a CAR is a cell surface receptor that comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain in a combination not naturally found in a single protein. The extracellular domain comprises an antigen-binding domain, which may be an antibody or antibody fragment. The antibody or antibody fragment may be a human antibody or fragment, a humanized antibody or fragment, or a non-human antibody or fragment. Typically, the antigen-binding domain is an antibody fragment such as a Fab or scFv. Most typically, the antigen-binding domain is an scFv. The extracellular domain typically further comprises a spacer (or hinge) region that links the antigen-binding domain to the transmembrane domain. The spacer region may be derived from an immunoglobulin, such as IgG1 or IgG4, or from alternative cell surface proteins, including, but not limited to, CD4, CD8, or CD28.

[0049] In certain embodiments, the bispecific polypeptides of the invention comprise a second binding domain derived from an anti-idiotypic antibody or antigen-binding fragment thereof, where the anti-idiotypic antibody is the anti-idiotypic antibody of the antibody portion of the CAR. In other words, the second binding domain binds to the antigen-binding domain of the CAR.

[0050] Anti-idiotypic antibodies are known in the art, and one of skill in the art would certainly be able to utilize the antigen-binding domains of these antibodies in designing the bispecific polypeptides of the invention. Thus, in some embodiments, the second binding domain of the bispecific polypeptides of the invention comprises an antibody or antibody fragment derived from an anti-idiotypic antibody that is specific for anti-CD19, anti-Her2, or other antibody, or an antigen-binding fragment of an anti-idiotypic antibody for binding to an antibody found in a CAR.

[0051] Many anti-idiotypic antibodies are known in the art. For example, WO 2014 / 190273 and Jena et al. 2013, PLoS One, 8(3):e57838 describe an anti-idiotypic antibody (mAb clone number 136.20.1) that recognizes anti-CD19^ scFv FMC63, which has been used in many CAR constructs in the current development.

[0052] In certain embodiments, the second binding domain of a bispecific polypeptide of the invention comprises an antibody or antigen-binding domain derived from an anti-idiotypic antibody specific for an anti-CD19 antibody, or an antibody or antigen-binding domain derived from an antigen-binding fragment of an anti-idiotypic antibody, which may have one or more of the same CDRs (i.e., one or more or all of VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3, using the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions) as the mAb clone of No. 136.20.1. In some embodiments, a multispecific antigen-binding construct comprises an antigen-binding polypeptide construct derived from an anti-idiotypic antibody specific for an anti-CD19 antibody, or an antigen-binding fragment of an anti-idiotypic antibody, which may have one or more (e.g., two) variable regions from the mAb clone of No. 136.20.1. In some embodiments, the multispecific antigen-binding construct comprises an antigen-binding polypeptide construct derived from an anti-idiotypic antibody that is specific for an anti-CD19 antibody or an antigen-binding fragment of an anti-idiotypic antibody that binds to the same epitope as the mAb clone No. 136.20.1.

[0053] Other examples of anti-idiotypic antibodies include commercially available anti-idiotypic antibodies from AbD Serotec®, anti-idiotypic antibodies specific for the anti-CD22 antibody described in WO 2013 / 188864, anti-idiotypic antibodies specific for the anti-CEA antibody described in WO 97 / 34636, anti-idiotypic antibodies specific for the anti-GD2 antibody described in U.S. Pat. No. 5,935,821, and anti-NY-ESO-1 antibody described in Jakka et al. 2013, Anticancer Research, 33(10):4189-420. Custom anti-idiotypic antibodies are also available from AbD Serotec®.

[0054] Alternatively, anti-idiotypic antibodies against CARs targeting CD19 or other tumor-associated antigens can be generated according to the methods described in Jena et al., supra, and used to construct anti-idiotypic antigen-binding polypeptide constructs.

[0055] In some embodiments, the bispecific polypeptide comprises a second binding domain that binds to an extracellular region of the CAR that is not involved in antigen binding. The second binding domain of the bispecific polypeptide of the invention can bind to any antigen on the extracellular domain of the CAR that is present on an immune cell. For example, the second binding domain of the bispecific polypeptide can bind to the hinge region of the CAR (i.e., between the transmembrane domain and the scFv portion of the CAR), or the spacer region between the variable heavy chain and variable light chain of the scFv portion of the CAR, or any other region of the CAR.

[0056] In some embodiments, the hinge region may be an scFv-CD28 or scFv-CD8 junction that includes a neoepitope that can be targeted by a second binding domain. In some embodiments, the hinge region may include a mutant (Fc binding null) IgG CH2 / 3 that can be targeted by a second binding domain. In some embodiments, the hinge region may include a spacer, such as the Strep tag II described by Liu et al. (2016, Nature Biotechnology, 34:430-434), that can be targeted by a second binding domain.

[0057] An example of an anti-CAR antibody that binds to the hinge region of a CAR molecule is the 2D3 antibody described in WO 2014 / 190273, which binds to the IgG4 CH2-CH3 hinge region. In some embodiments, the multispecific antigen-binding construct comprises an antigen-binding polypeptide construct that binds to the IgG4 CH2-CH3 hinge region. In some embodiments, the multispecific antigen-binding construct comprises an antigen-binding polypeptide construct that binds to the IgG4 CH2-CH3 hinge region and has one or more of the same CDRs as 2D3 (i.e., one or more or all of VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3), or has one or more (e.g., two) variable regions of 2D3 described in WO 2014 / 190273. In some embodiments, the second binding domain of the bispecific polypeptide binds to the IgG4 CH2-CH3 hinge region and binds to the same epitope as 2D3 described in WO 2014 / 190273.

[0058] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide comprising an antigen-binding domain linked to at least one intracellular signaling domain. The antigen-binding domain of a CAR is the functional portion of the CAR that specifically binds to (i.e., specifically targets) an antigen expressed on a cancer cell (i.e., a "tumor-associated antigen"). Examples of tumor-associated antigens are known to those skilled in the art, and specific examples thereof include Her2, CEA, FBP, CD19, and CD209.

[0059] As used herein, "tumor-associated antigen" refers to an antigen expressed by cancer cells. Tumor-associated antigens may or may not be expressed by non-tumor cells. If a tumor-associated antigen is not expressed by non-tumor cells (i.e., unique to tumor cells), it can also be called a "tumor-specific antigen." If a tumor-associated antigen is not unique to tumor cells, it may also be expressed on non-tumor cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of the antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A tumor-associated antigen may be an antigen expressed on non-tumor cells during fetal development, when the immune system is immature and unable to respond, or it may be an antigen that is normally present at low levels on normal cells but expressed at much higher levels on tumor cells. Those tumor-associated antigens of greatest clinical interest are differentially expressed compared to corresponding non-tumor tissues, allowing preferential recognition of tumor cells by specific T cells or immunoglobulins.

[0060] Full and sustained T cell activation and proliferation requires a primary initiation signal (signal 1), a secondary costimulatory receptor-engaging signal (signal 2), and a cytokine receptor-engaging signal (signal 3). Because CAR T cells do not operate in an MHC-restricted manner, their interactions with APCs are generally defective, and signals 2 and 3 are severely attenuated. Thus, the incorporation of one or more signaling domains can profoundly affect the level and maintenance of T cell activation in response to tumor-associated antigens, which can result in increased cytokine production.

[0061] In one embodiment, the CAR comprises at least one intracellular signaling domain. Suitable signaling domains are familiar to those skilled in the art, and specific examples include CD3ζ, CD28, 41BB, DAP10, OX40, ICOS, DAP12, KIR2DS2, 4-1BB, CD3ε, CD35, CD3ν, CD25, CD27, CD79A, CD79B, CARD11, FcRa, Fcftp, FcRy, Fyn, HVEM, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, SLAMF1, Slip76, pTa, TCRa, TCRβ, TRIM, Zap70, PTCH2, and LIGHT. In one embodiment, the CAR comprises at least two intracellular signaling domains. In another embodiment, the intracellular signaling domain of the CAR comprises CD28 and CD3ζ signaling domains.

[0062] In another embodiment, the second binding domain binds to an antigen on an immune cell expressing a CAR, where the antigen is not the CAR or is not present on the CAR. In other words, the immune cell expressing a CAR may further comprise one or more antigens that can be bound by the second binding domain of the bispecific polypeptide of the invention. For example, the antigen may include a naturally occurring protein present on the surface of the immune cell (such as CD3, CD4, CD8, CD25, CD127, CD196 (CCR6), CD27, CD28, CD45RA, CD45RO, CD62L, CD197, and HLA-DR). Alternatively, the antigen on the immune cell may be an artificially introduced antigen, such as an affinity tag. Examples of peptide tags that can be present on the surface of immune cells include peptide tags (e.g., FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, Strep tag, eXact tag) and protein tags (e.g., GST tag, MBP tag, GFP tag).

[0063] The first and second binding domains of the bispecific polypeptides of the invention are preferably in the form of antibodies or antibody fragments. As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains. Further disclosed herein are antigen-binding fragments, mutants, variants, and derivatives thereof that retain the essential epitope-binding function of an antibody molecule. Such mutants, variants, and derivatives are known to those of skill in the art, and specific examples thereof are described elsewhere herein.

[0064] An antibody heavy chain will typically comprise a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region will typically comprise three domains, CH1, CH2, and CH3. A light chain will typically comprise a light chain variable region (LCVR or VL) and a light chain constant region CL. The VH and VL regions can be further subdivided into regions of hypervariability, also known as complementarity-determining regions (CDRs), separated by framework regions (FR). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0065] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. Specific examples of antigen-binding fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment or F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a single-chain variable fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment containing a single variable domain (Ward et al. 1989, Nature, 341:544-6); and (vi) an isolated CDR.

[0066] In one embodiment, the first binding domain comprises an scFv. In another embodiment, the second binding domain comprises an scFv.

[0067] Suitable immune cells are familiar to those skilled in the art, and specific examples thereof include T cells, tumor-infiltrating lymphocytes (TILs), and natural killer (NK) cells. In one embodiment, the cells are T cells, natural killer (NK) cells, or tumor-infiltrating lymphocytes (TILs).

[0068] In one preferred embodiment, the CAR-expressing immune cells are T cells. Specific examples of suitable T cells include helper T cells (HTL; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells may be T cells or any other subset of T cells. Other specific examples of suitable T cells include T cells that express one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.

[0069] Sources of cells for use in accordance with the present invention are known to those skilled in the art, and specific examples thereof include peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, the cells are derived from whole blood.

[0070] As used herein, the term "sequence identity" or "sequence homology" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules, or between two polypeptide molecules. Two molecules are homologous, or identical, at a subunit position in both molecules if that position is occupied by the same monomer subunit, e.g., if one position in each of the two DNA molecules is occupied by adenine. The homology between two sequences is a linear function of the number of matching or homologous positions; e.g., if half the positions in two sequences (e.g., five positions in a polymer 10 subunits in length) are homologous, the two sequences are 50% homologous; if 90% of the positions (i.e., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0071] In one embodiment the bispecific polypeptide comprises a first binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or 4 or an amino acid sequence having at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or 4.

[0072] In one embodiment the bispecific polypeptide comprises a second binding domain comprising the amino acid sequence of SEQ ID NO: 6 or 8 or an amino acid sequence having at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 6 or 8.

[0073] In one embodiment the bispecific polypeptide comprises amino acids comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 18, 20 or 22 or an amino acid sequence having at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to an amino acid selected from the group consisting of SEQ ID NO: 16, 18, 20 or 22.

[0074] In one embodiment the bispecific polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 2. a first binding domain; and a second binding domain comprising an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 6.

[0075] In one embodiment the bispecific polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 2. a first binding domain; and a second binding domain comprising an amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO:8.

[0076] In one embodiment the bispecific polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 4. a first binding domain; and a second binding domain comprising an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 6.

[0077] In one embodiment the bispecific polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 4. a first binding domain; and a second binding domain comprising an amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO:8.

[0078] Bispecific polypeptides of the invention may comprise a linker sequence linking the first and second binding domains. Linkers may, for example, function to link two domains of an antigen-binding polypeptide construct (such as the VH and VL of an scFv or diabody), or they may function to link two antigen-binding polypeptide constructs together (such as two or more Fabs or sdAbs), or they may function to link an antigen-binding polypeptide construct to a scaffold. In some embodiments, a bispecific polypeptide may comprise multiple (i.e., two or more) linkers; for example, one or more scFvs linked to a scaffold may comprise a linker connecting the VH and VL of the scFv and a linker connecting the scFv to the scaffold. Suitable linkers are known in the art and can be readily selected by one of skill in the art based on the intended use of the linker (see, e.g., Mueller & Kontermann, "Bispecific Antibodies" in Handbook of Therapeutic Antibodies, Wiley-VCH Verlag GmbH & Co. 2014).

[0079] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art and contain glycine and serine units arranged in various orders. Examples include (GS), (GSGGS), n , (GGGS) n and (GGGGS) n (In the formula, n is at least 1, typically an integer between 1 and about 10, for example, an integer between 1 and about 8, 1 and about 6, or about 1 and about 5, but is not limited to these.

[0080] Other useful linkers include sequences derived from immunoglobulin hinge sequences. The linker can include all or part of a hinge sequence from any one of the four IgG classes, and can optionally include additional sequences. For example, the linker can include an immunoglobulin hinge sequence and a portion of a glycine-serine sequence. A non-limiting example is a linker that includes the first 15 residues of an IgG1 hinge, followed by a GlySer linker sequence about 10 amino acids in length, as described above.

[0081] The length of the linker will vary based on its intended use. An appropriate linker length can be readily selected by one skilled in the art. For example, if the linker must connect the VH and VL domains of an scFv, the linker is typically about 5 to about 20 amino acids in length, e.g., about 10 to about 20 amino acids, or about 15 to about 20 amino acids in length. If the linker must connect the VH and VL domains of a diabody, the linker must be sufficiently short to prevent binding of these two domains within the same chain. For example, the linker can be about 2 to about 12 amino acids in length, e.g., about 3 to about 10 amino acids in length, or about 5 amino acids in length.

[0082] In some embodiments, when a linker must connect two Fab fragments, the linker can be selected to maintain the relative spatial structure of the paratopes of the F(ab') fragments and to form a covalent bond equivalent to the disulfide bond within the core hinge of IgG. In this context, suitable linkers include, for example, IgG hinge regions, such as those from IgG1, IgG2, or IgG4. Modified versions of these exemplary linkers can also be used. For example, modifications to improve the stability of IgG4 hinges are known in the art (see, e.g., Labrijn et al. 2009, Nature Biotechnology, 27:767-771).

[0083] The linker may comprise a sequence of amino acid residues connecting the first and second binding domains. Alternatively, the first and second binding domains may be linked by chemical bonding (e.g., to form a bis-aryl bond between the domains). Examples of suitable methods for chemically bonding binding domains are known in the art. Such methods include the use of succinimidyl compound modification of primary amines present on lysine residues, as used in TriLink Technologies' bioconjugation reagents. In one embodiment, when the first and / or second binding domains are scFvs, the bispecific polypeptide comprises a linker sequence between the VH and VL domains of the scFv. Suitable linker sequences will be known to those skilled in the art, and specific examples thereof include fairly flexible and hydrophilic amino acid residues. In one embodiment the linker sequence is selected from the group consisting of SEQ ID NO: 10, 12 or 14 or an amino acid sequence having at least 70% sequence identity, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity to SEQ ID NO: 10, 12 or 14.

[0084] The antibody or antibody fragment may contain additional amino acids or molecules for purification or identification. For example, the antibody may contain an epitope or affinity tag. Specific examples of such epitope or affinity tags include peptide tags (e.g., FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, Strep tag, eXact tag) and protein tags (e.g., GST tag, MBP tag, GFP tag). In one embodiment, the epitope or affinity tag is a His tag.

[0085] Nucleic acids and vectors In another aspect disclosed herein, there is provided a nucleic acid encoding the bispecific polypeptide described herein. In yet another aspect, there is provided a cell comprising the vector described herein.

[0086] The terms "polynucleotide," "polynucleotide sequence," "nucleotide sequence," "nucleic acid," or "nucleic acid sequence" are used interchangeably herein to designate mRNA, RNA, cRNA, cDNA, or DNA. The term refers to a polymeric form of nucleotides, typically at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of any type of nucleotide. The term includes single- and double-stranded forms of RNA and DNA.

[0087] As used herein, the term "gene" includes a nucleic acid molecule that can be used to produce mRNA, optionally with additional elements to aid in this process. A gene may or may not be usable to produce a functional protein. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions).

[0088] As used herein, "transgene" is used to describe genetic material that has been or is to be artificially introduced into the genome of a host organism and that is inherited by the host's progeny. In some embodiments, the transgene confers a desired characteristic or otherwise results in a desired therapeutic outcome on a T cell into which it is introduced.

[0089] As used herein, the terms "encode," "encoding," and the like refer to the ability of a nucleic acid to provide another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" if it can be transcribed and / or translated to produce a polypeptide, or if it must be processed into a form that can be transcribed and / or translated to produce a polypeptide. Such a nucleic acid sequence can include both coding and non-coding sequences. Thus, the terms "encode," "encoding," and the like include an RNA product resulting from the transcription of a DNA molecule, a protein resulting from the translation of an RNA molecule, a protein resulting from the transcription of a DNA molecule to produce an RNA product and subsequent translation of the RNA product, or a protein resulting from the transcription of a DNA molecule to provide an RNA product, processing the RNA product to provide a processed RNA product (e.g., mRNA), and subsequent translation of the processed RNA product.

[0090] In one embodiment, the nucleic acid sequence encoding the bispecific polypeptide is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15, 17, 19, 21, 31 or 33, or has at least 70% sequence identity to SEQ ID NO: 15, 17, 19, 21, 31 or 33, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81% , preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or more preferably at least 99% sequence identity.

[0091] In another aspect, a vector is provided comprising a nucleic acid described herein operably linked to a control sequence.

[0092] The term "control element" or "control sequence" refers to a nucleic acid sequence (e.g., DNA) required for the expression of an operably linked coding sequence in a particular cell. Control sequences suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or tail sequences controlling mRNA stability, and sequences that target the product encoded by the transcribed polynucleotide to an intracellular compartment within the cell or to the extracellular environment.

[0093] Typically, regulatory sequences include, but are not limited to, nucleic acid sequences encoding promoter sequences, 5' non-coding regions, cis-regulatory regions such as functional binding sites for transcriptional or translational regulatory proteins, upstream open reading frames, ribosomal binding sequences, transcription initiation sites, translational initiation sites and / or leader sequences, stop codons, translational stop sites, and 3' non-translated regions. Constitutive or inducible promoters known in the art are contemplated. The promoters may be either naturally occurring promoters or hybrid promoters that combine elements of more than one promoter.

[0094] The contemplated promoter sequence may be native to mammalian cells or may be derived from another source whose region is functional in the selected organism. The choice of promoter will vary depending on the intended host cell. For example, promoters that can be used for expression in mammalian cells include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, the β-actin promoter, and viral promoters such as the SV40 large T antigen promoter, the human cytomegalovirus (CMV) immediate early (IE) promoter, the Rous sarcoma virus long terminal repeat (LTR) promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus promoter, and the HPV promoter, particularly the HPV upstream regulatory region (URR). All of these promoters have been clearly described in the art and are readily available.

[0095] Enhancer elements can be used herein to increase the expression level of nucleic acid sequences in vector constructs. Examples include the SV40 early gene enhancer described in Dijkema et al. (1985, EMBO Journal, 4:761), the enhancer / promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus described, for example, in Gorman et al. (1982, Proceedings of the National Academy of Science, USA, 79:6777), and elements derived from human CMV, such as those contained in the CMV intron A sequence described, for example, in Boshart et al. (1985, Cell, 41:521).

[0096] The vector construct may further comprise a 3' non-translated sequence. The 3' non-translated sequence refers to that portion of a gene that includes a DNA segment containing a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is characterized by the addition of a polyadenylic acid tract to the 3' end of a pre-mRNA. Polyadenylation signals are generally recognized by the presence of homology to the canonical form 5'AATAAA-3', although variations are not uncommon. The 3' non-translated regulatory DNA sequence preferably comprises approximately 50 to 1,000 nt and may contain transcription and translation termination sequences in addition to the polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.

[0097] As used herein, the terms "operably connected" or "operably linked" refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence refers to the positioning and / or orientation of the control sequence relative to the coding sequence that permits expression of the coding sequence under conditions compatible with the control sequences.

[0098] As used herein, the terms "open reading frame" and "ORF" are used interchangeably herein to refer to the amino acid sequence encoded between the translation initiation and termination codons of a coding sequence. The terms "initiation codon" (e.g., ATG) and "termination codon" (e.g., TGA, TAA, TAG) refer to units of three adjacent nucleotides ("codons") within a coding sequence that specify the initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0099] As used herein, the term "recombinant" as applied to "nucleic acid molecule," "polynucleotide," etc., is understood to mean an artificial nucleic acid structure (i.e., a non-replicating cDNA or RNA; or a replicon, a self-replicating cDNA or RNA) that can be transcribed and / or translated in a cell as described herein.

[0100] The recombinant nucleic acid molecule or polynucleotide can be inserted into a vector. Non-viral vectors, such as plasmid expression vectors, or viral vectors can be used. Types of vectors and techniques for inserting nucleic acid constructs according to the present invention are known in the art, and specific examples include cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0101] In one embodiment, the vector comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26.

[0102] The nucleic acid sequences, polynucleotides or vector constructs described herein include heterologous sequences that do not occur in nature.

[0103] In yet another aspect, there is provided a host cell comprising the vector described herein. Suitable host cells will be known to those of skill in the art, and specific examples thereof include bacterial cells (e.g., E. coli, P. mirabilis), fungal cells (e.g., S. cerevisiae, P. pastoria, T. reesei), plant cells, insect cells (e.g., SF-9, SF21, Hi-5), or mammalian cells. In one embodiment, the host cell is a mammalian cell. Suitable mammalian cells will be known to those of skill in the art, and specific examples thereof include CHO or 293T cells. These cells are widely available from commercial suppliers.

[0104] Methods for producing bispecific polypeptides In another aspect, there is provided a method for producing a bispecific polypeptide described herein, the method comprising: (i) culturing a cell described herein in a culture medium and under conditions suitable for expression of the bispecific polypeptide; and (i) isolating the bispecific polypeptide from the cell or culture medium.

[0105] The bispecific polypeptides described herein can be produced using any number of expression systems that will be known to those of skill in the art, specific examples of which include production in or by bacteria (e.g., E. coli, P. mirabilis), fungi (e.g., S. cerevisiae, P. pastoria, T. reesei), plants or plant cells, insects or insect cells (e.g., SF-9, SF21, Hi-5), or mammalian cells. In one embodiment, the expression system is a mammalian expression system. Suitable mammalian expression systems will be known to those of skill in the art, specific examples of which include CHO or 293T expression systems. These expression systems are widely available from commercial suppliers. In one embodiment, the bispecific polypeptides are produced using a mammalian expression system.

[0106] In another aspect, there is provided a method for producing a bispecific polypeptide as described herein, the method comprising combining a first binding domain and a second binding domain under conditions suitable to produce a chemically conjugated bispecific polypeptide comprising the first binding domain and the second binding domain.

[0107] In one preferred embodiment, the chemically conjugated bispecific polypeptide is formed after modification of primary amines present on lysine residues with succinidyl compounds, as described elsewhere herein.

[0108] It would be within the skill of one in the art to utilize commercially available antibodies or their antigen-binding domains to generate the bispecific polypeptides described herein. Furthermore, it would be within the ability of one in the art to replicate an antibody or antigen-binding domain based on published sequence information for an antibody or antigen-binding domain with the desired specificity and thereby include such an antibody or antigen-binding domain in a bispecific polypeptide in accordance with the present invention. In particular, it will be understood that the present invention encompasses the generation of any bispecific antibody that has been engineered to have the specific binding affinity of the first and second binding domains described herein (i.e., a first binding domain for binding to an antigen on an antigen-presenting cell, preferably a professional antigen-presenting cell such as a dendritic cell, and a second binding domain for binding to an antigen on an immune cell expressing a CAR, including when the antigen is an antigen on a CAR). Those skilled in the art will understand that any combination of antigen-binding domains and desired binding specificities can be utilized to obtain the bispecific polypeptides of the present invention.

[0109] Pharmaceutical Composition In another aspect, there is provided a pharmaceutical composition comprising a bispecific polypeptide described herein and a pharmaceutically acceptable carrier.

[0110] The compositions described herein can be prepared by methods known in the art and are suitable for parenteral administration to mammals, particularly humans, and comprise a therapeutically effective amount of the composition with one or more pharmaceutically acceptable carriers or diluents.

[0111] As used herein, the term "pharmaceutically acceptable carrier" refers to a suitable carrier, diluent, or excipient. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, and solutes that render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorbing agents, etc. The compositions of the present invention may also contain other additional physiologically active agents.

[0112] Carriers are typically pharmaceutically "acceptable" in the sense that they are compatible with other ingredients in the composition and are not harmful to the subject.Compositions include compositions suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration.The compositions can conveniently be presented in unit dosage form and can be prepared by any method well known in the field of pharmacy.Such methods include preparing a carrier for binding with isolated T cells.Generally, compositions are prepared by uniformly and intimately combining any active ingredient with a liquid carrier.

[0113] In one embodiment, the composition is suitable for parenteral administration, hi another embodiment, the composition is suitable for intravenous administration.

[0114] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bactericides, and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0115] The present disclosure also contemplates the combination of the compositions described herein with other active agents and / or in addition to other treatment regimens or modalities, such as radiation therapy or surgery. When the compositions described herein are used in combination with a known active agent, the combination can be administered either sequentially (either continuously or with periods of no treatment), simultaneously, or as an admixture. Suitable anti-cancer agents will be known to those skilled in the art. Combination treatments, in which a composition of the present invention is followed by a known treatment, or treatment with a known agent is followed by treatment with a composition of the present invention as maintenance therapy, are also contemplated. For example, in the treatment of cancer, the compositions of the present invention can be administered in combination with alkylating agents (platinum-containing alkylating agents such as mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin or cisplatin, carboplatin, and oxiplatin), antimetabolites (purine or pyrimidine analogs or antifolates such as azathioprine and mercaptopurine), anthracyclines (daunorubicin, doxorubicin, epirubicin, idarubicin, barbicin, mitoxantrone, or anthracycline analogs), plant alkaloids ( Vinca alkaloids or taxanes such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel or doestaxel), topoisomerase inhibitors (type I or type II topoisomerase inhibitors, etc.), podophyllotoxins (etoposide or teniposide, etc.), tyrosine kinase inhibitors (imatinib mesylate, nilotinib or dasatinib, etc.), adenosine receptor inhibitors (A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP or A2 BIt is contemplated that the therapeutic agent may be administered in combination with an anti-PD-1 inhibitor (e.g., PD-1 R inhibitors, PSB-1115), an adenosine receptor agonist (e.g., CCPA, IB-MECA, and CI-IB-MECA), a checkpoint inhibitor of the PDL-1:PD-1 axis, including checkpoint inhibitors nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A, or MSB0010718C), an inhibitor of the CTLA-4 pathway (e.g., ipilimumab and tremelimumab), an inhibitor of the TIM-3 pathway, or an agonist monoclonal antibody known to promote T cell function (including anti-OX40, such as MEDI6469; and anti-BB, such as PF-05082566).

[0116] In yet another aspect, the present invention provides kits or articles of manufacture comprising one or more bispecific polypeptides of the invention, nucleic acids encoding said bispecific polypeptides and / or pharmaceutical compositions as described above.

[0117] In yet another aspect, there is provided a kit for use in the above-described therapeutic applications, comprising: (a) a container holding a bispecific polypeptide, nucleic acid, vector, or pharmaceutical composition of the invention; and (b) The kit is provided with a label or package insert with instructions for use.

[0118] The kit may further comprise one or more active principles or ingredients for treating cancer. For example, the kit may further comprise immune cells expressing a CAR.

[0119] The "kit" or "article of manufacture" may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a therapeutic composition that is effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used for treating the condition of choice. In one embodiment, the label or package insert includes instructions for use indicating that the therapeutic or prophylactic composition can be used to treat cancer or other conditions described herein.

[0120] The kit may include a second container having (a) a therapeutic or prophylactic composition; and (b) a second active ingredient contained therein. Kits according to embodiments of the invention may further include a package insert indicating that the composition and other active ingredients can be used to treat a cancer or condition described herein. Alternatively, or in addition, the kit may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, suitable examples of which include other buffers, diluents, filters, needles, and syringes, as would be known to those of skill in the art.

[0121] Treatment method In another aspect, provided is a method for treating cancer, comprising co-administering to a subject in need thereof therapeutically effective amounts of: (i) immune cells expressing a CAR; and (ii) a bispecific polypeptide or pharmaceutical composition described herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the subject's endogenous APCs and to an antigen on the CAR expressed by the immune cells in vivo to stimulate immune cell activation and proliferation to treat the cancer.

[0122] Co-administration of a CAR-expressing immune cell with a bispecific polypeptide or pharmaceutical composition described herein can be achieved by formulating the immune cell and the bispecific polypeptide or pharmaceutical composition in the same composition (e.g., for simultaneous co-administration) or by formulating them as different compositions for sequential administration. "Sequential" administration means that there is an interval between the administration of the immune cell and the bispecific polypeptide or pharmaceutical composition. The interval between sequential administrations can be a few seconds, minutes, hours, or days. In one embodiment, periodic re-administration of the immune cell and the bispecific polypeptide or pharmaceutical composition may be required to achieve the desired therapeutic effect. Sequential administration can be in any order.

[0123] The inventors have surprisingly demonstrated that the bispecific polypeptides described herein stimulate the activation and proliferation of immune cells in vivo, thereby improving the efficacy of immune cell therapy, such as CAR T cell therapy. Accordingly, the bispecific polypeptides can be referred to as "adjuvants." As used herein, the term "adjuvant" refers to a bispecific polypeptide that can increase the magnitude of the immune response elicited by immune cells expressing a CAR beyond the magnitude of the immune response that would be expected from immune cells expressing the CAR alone.

[0124] Activation of immune cells can be accomplished by providing a primary stimulatory signal, for example, through stimulation of the T cell TCR / CD3 complex or CD2 surface protein, and providing a second costimulatory signal through an accessory molecule, such as CD28 or 4-1BBL. In addition to the primary stimulatory signal provided through the TCR / CD3 complex or via CD2, a second costimulatory signal is required to induce a T cell response. In certain embodiments, a CD28-binding agent can be used to provide the costimulatory signal. Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind B7-H3.

[0125] The terms "expanded," "proliferation," "expanding," "propagation," and the like are used interchangeably herein to refer to an increase in the number of immune cells in the preparation of a pharmaceutical composition either before or after administration of the cells to a subject. Immune cells can be expanded using any cell culture method known in the art. For example, immune cells can be expanded in in vitro tissue culture systems, including liquid culture, monolayers, and the like.

[0126] Therapeutic regimens for treating cancer can be determined by those skilled in the art, but will typically depend on factors including, but not limited to, the age, weight, and general health of the subject, as well as the type, size, stage, and receptor status of the tumor. Another determining factor may be the risk of developing recurrent disease. For example, a subject at high risk or higher risk of developing recurrent disease, or who has developed recurrent disease, may be prescribed a more aggressive treatment regimen compared to a subject at low risk or lower risk of developing recurrent disease. Similarly, a subject identified as having a more advanced stage of cancer, such as stage III or IV, may be prescribed a more aggressive treatment regimen compared to a subject with a less aggressive stage of cancer.

[0127] As used herein, the term "cancer" refers to any condition associated with abnormal cell proliferation. Such conditions are known to those of skill in the art. In one embodiment, the cancer is a solid cancer. In another embodiment, the cancer is a Her2-positive cancer. In another embodiment, the cancer is selected from the group consisting of breast cancer, prostate cancer, and lung cancer.

[0128] As used herein, the terms "treat," "treatment," and "treating" refer to any and all uses that in any way prevent, hinder, delay, negate, or reverse the onset or progression of a cancerous condition or symptom, or otherwise cancer or other undesirable symptom. Accordingly, the terms "treating" and the like can be considered in their very widest possible context. For example, treatment does not necessarily mean that a subject is treated until complete recovery or cure. In conditions presenting or characterized by multiple symptoms, treatment does not necessarily relieve, prevent, hinder, delay, negate, or reverse all of the symptoms, but may relieve, prevent, hinder, delay, negate, or reverse one or more of the symptoms.

[0129] The subject whose cancer needs to be treated may be a human or a mammal of economic and / or social importance to humans, for example, non-human carnivores (e.g., cats and dogs), pigs (e.g., pigs, domestic pigs and wild boars), ruminants (e.g., cattle, oxen, sheep, giraffes, deer, goats, bison and camels), horses, and birds and poultry, including endangered zoo bird species, and more particularly domesticated poultry, such as turkeys, chickens, ducks, geese, guinea fowl, etc. The term "subject" does not denote a particular age. Thus, it is intended to include adult, juvenile and newborn subjects.

[0130] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to any subject to which the present disclosure is applicable. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0131] As used herein, the term "therapeutically effective amount" means an amount of cells that is sufficient to treat cancer when administered to a mammal, particularly a human, in need of such treatment. The exact amount of modified cells to be administered can be determined by a physician, taking into account individual differences in the subject's age, weight, tumor size, extent and condition of infection or metastasis.

[0132] Typically, the administration of immune cell (e.g., CAR-T cell) therapy is defined by the number of cells per kg of body weight. However, since the modified cells will replicate and proliferate after introduction, the administered cell dose will not resemble the final steady-state number of cells. In one embodiment, a pharmaceutical composition comprising the modified cells of the present invention is administered in a dose of 10 cells. 4 ~10 9 In another embodiment, the pharmaceutical compositions comprising the modified cells of the present invention can be administered at a dose of 10 cells / kg body weight, including all integer values ​​within those ranges. 5 ~10 6 It can be administered at a dose of 100 mg / kg (body weight).

[0133] Compositions comprising the bispecific polypeptides described herein can be administered multiple times at these doses. Optimal dosages and treatment regimens for a particular subject can be readily determined by one of skill in the art by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0134] In one embodiment, the immune cells are derived from autologous cells. In another embodiment, the immune cells are derived from allogeneic cells.

[0135] The term "autologous" refers to any material derived from the same individual that is to be subsequently reintroduced into that individual.

[0136] The term "allogeneic" refers to any material derived from a different individual of the same species as the individual into whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material derived from individuals of the same species is sufficiently genetically distinct to interact antigenically.

[0137] Cell manufacturing To achieve a sufficient therapeutic dose of an immune cell composition, methods for generating immune cells typically involve one or more rounds of stimulation, activation, and / or expansion. According to the methods disclosed herein, immune cells can be stimulated to activate and expand in vivo and in vitro.

[0138] Accordingly, in another aspect disclosed herein, there is provided a method for stimulating immune cell activation and proliferation in vivo, comprising co-administering to a subject effective amounts of: (i) immune cells expressing a CAR; and (ii) a bispecific polypeptide or pharmaceutical composition described herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the subject's endogenous APCs and to an antigen on the CAR expressed by the immune cells in vivo.

[0139] The in vivo activation and expansion of immune cells described herein facilitates a reduction in the number of immune cells required for each therapeutic dose of immune cells.

[0140] In another aspect, provided is a method for stimulating the activation and proliferation of immune cells in vitro, comprising culturing an isolated immune cell expressing a CAR in a culture medium comprising: (i) an APC or an APC mimetic, and (ii) a bispecific polypeptide disclosed herein, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the APC or APC mimetic and to an antigen on the CAR expressed by the immune cell.

[0141] The immune cell generation method herein contemplates simple and robust culture initiation and activation steps that contribute to the resulting immune cell composition being an excellent therapeutic product. In one embodiment, culture initiation and activation includes seeding a cell population in a cell culture vessel, e.g., a cell culture bag, a GREX bioreactor, a WAVE bioreactor, etc., and activating the immune cells through primary and costimulatory immune cell signaling pathways. The cell composition may be further cultured in the presence of one or more additional growth factors or cytokines, e.g., IL-2, IL-7, and / or IL-15, or any suitable combination thereof.

[0142] In another embodiment, activating and expanding immune cells comprises culturing isolated immune cells with APCs or APC mimics, which in one embodiment are selected from the group consisting of donor-derived APCs, synthetic artificial APCs (aAPCs), microbeads functionalized with activating antibodies for CD3 and CD8 (Dynabeads), autologous monocyte-derived dendritic cells (mo-DCs), and scaffolds that mimic APCs.

[0143] In certain embodiments, initiation of the culture begins with culturing a population of cells, e.g., PBMCs, comprising T cells at a desired density, e.g., 1-5 x 10 cells, in a cell culture vessel in a suitable cell culture medium containing, e.g., one or more additional cytokines, a primary stimulatory ligand, and a costimulatory ligand. 6 In another embodiment, cytokines, stimulatory and costimulatory ligands can be subsequently added to the PBMCs in cell culture medium.

[0144] In one embodiment, the cell culture vessel is a cell culture bag, including but not limited to MACS® GMP cell proliferation bags, MACS® GMP cell differentiation bags, EXP-Pak™ cell proliferation biocontainers, VueLife™ bags, KryoSure™ bags, KryoVue™ bags, Lifecell® bags, PermaLife™ bags, X-Fold™ bags, Si-Culture™ bags, and VectraCell™ bags, as discussed elsewhere herein.

[0145] In certain embodiments, cells are seeded in a cell culture vessel containing a suitable cell culture medium. Specific examples of suitable cell culture media include, but are not limited to, TCGM supplemented with amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts to grow and expand immune cells; X-VIVO™ 15 supplemented with 2 mM GlutaMAX™-I, 10 mM HEPES, and 5% human AB serum, CTS™ OpTmizer™ T Cell Expansion SFM (Life Technologies), CTS™ AIM V® Medium (Life Technologies), RPMI 1640, Clicks, DMEM, MEM, α-MEM, F-12, X-Vivo 15 (Lonza), CellGro® Serum-Free Medium (CellGenix), and X-Vivo 20 (Lonza).

[0146] Cell culture media contemplated herein may further comprise one or more factors, including, but not limited to, serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α.

[0147] In one embodiment, the APC or APC mimic is selected from the group consisting of donor-derived APC, synthetic artificial APC (aAPC), microbeads (Dynabeads) functionalized with activating antibodies for CD3 and CD8, autologous monocyte-derived dendritic cells (mo-DC), and scaffolds that mimic APC.

[0148] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within the spirit and scope of the invention. The invention further includes all steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of two or more of said steps or features.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0150] The various embodiments enabled herein are further described by the following non-limiting examples. [Example]

[0151] A. Material cell culture E0771 is a murine breast cancer cell line (Johnstone et al., 2015, Disease Models & Mechanisms, 8:237-51), 24JK is a methylcholanthrene-induced fibrosarcoma (Shiloni et al., 1993, Cancer Immunology and Immunotherapy, 37:286-92), and MC38 is a chemically induced colon adenocarcinoma (Corbett et al., 1975, Cancer Research, 35:2434-9). These cell lines originated from C57BL / 6 mice, and the parental cell lines were retrovirally transfected to express human Her2 antigen under the control of the murine stem cell virus long terminal repeat (LTR) promoter. These Her2+ cell lines are designated 24JK-Her2, MC38-Her2, and E0771-Her2. MDA-MB-231, PANC1 and A549 cell lines are Her2+.

[0152] All cell lines were maintained at 37°C in 5% CO in RPMI medium containing supplements including 10% heat-inactivated fetal bovine serum (FBS), 1 mmol / L sodium pyruvate, 2 mmol / L glutamine, 0.1 mmol / L non-essential amino acids, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U / mL penicillin, and 100 μg / mL streptomycin. E0771-Her2 was cultured in supplemented DMEM, which enhanced attachment to the flask.

[0153] CAR T cells PBMCs were collected from buffy coats of whole blood collected from human or mouse donors. Transduction of human T cells with Her2-specific CAR retroviral vectors or control empty vectors was performed according to the method described by Ritchie et al. (2013, Molecular Therapy, 21(11):2122-29). Transduction of mouse T cells with Her2-specific CAR retroviral vectors or control empty vectors was performed according to the method described by Mardiana et al. (2017, Cancer Research).

[0154] Other immune cells APCs were generated using irradiated PBMCs, FACS-sorted CD19+ B cells from PBMCs, or monocyte-derived dendritic cells (mo-DCs). Mo-DCs were generated in a mixture of immature and mature mo-DCs using a commercially available kit (Miltenyi Biotec).

[0155] B. BEAT Design BEAT was designed by linking two antibody scFv domains into a single polypeptide chain using a flexible linker. The first scFv domain was designed to specifically bind to CD40 (SEQ ID NO: 2) or MHCII (SEQ ID NO: 4). cDNA was isolated from hybridoma cell lines producing monoclonal antibodies against CD40 (FGK45) and MHCII (M5 / 114). Genes encoding the variable H and L domains of these monoclonal antibodies were cloned and genetically fused via a (G4S)3-linker sequence to generate APC-specific scFvs. The second scFv domain was designed to specifically bind to CD3 (SEQ ID NO: 6) or the c-myc tag (SEQ ID NO: 8) of the Her2-specific CAR described above, and CAR-specific scFvs were generated using the same method.

[0156] The APC-specific scFv and the CAR-specific scFv were recombinantly linked to generate a gene encoding BEAT (SEQ ID NOs: 17 and 19).

[0157] C. BEAT Generation The BEAT coding sequence was cloned into the pCDNA3.4 TOPO vector, which contains a cytomegalovirus (CMV) promoter, for high-level expression of the native protein in mammalian cell culture. The coding sequence of the insert was verified according to standard protocols. The Expi293 expression system (Invitrogen), jetPEI (Polyplus-transfection SA), and Lipofectamine were used to transfect mammalian cells for rapid and ultra-high-yield protein production of BEAT.

[0158] The Gateway cloning system (Invitrogen) was used for bacterial production of BEAT. Thus, the BEAT coding sequence was inserted into the entry plasmid to generate pDONR221ZeoBEAT. Competent E. coli was then transformed, and positive clones containing pDONR221ZeoBEAT were selected and sequenced. pDONR221ZeoBEAT was then combined with the pDestination vector pET15bGW to generate pET15bBEAT. Competent E. coli was then transformed, and positive clones containing pET15bBEAT were selected and sequenced. pET15bBEAT was then transformed into BL21DE3 E. coli and induced with IPTG to produce the BEAT polypeptide.

[0159] BEAT polypeptides containing His tags were collected using His60 Ni Superflow resin / Nickel beads, and the purity and concentration of BEATs were tested using the 6His Check Kit (Cisbio) electrophoresis and Western blot.

[0160] To confirm the binding of the recombinant BEAT polypeptide, the BEAT will be conjugated with a fluorescent dye. Binding of the labeled BEAT will be confirmed using flow cytometry. Furthermore, we will use competitive binding assays to confirm that the BEAT and the original monoclonal antibody bind to the same molecule.

[0161] Chemically conjugated BEAT polypeptides were prepared using a protein-protein conjugation kit (TriLink Bio Technologies). Briefly, antibodies specific for the myc tag (present in CAR), CD40, PD-L2, or Clec9a were desalted and concentrated into modification buffer using a Zebra column.

[0162] To prepare BEAT polypeptides, one antibody was modified with S-HyNic and the other with S-4FB, both at a molar substitution ratio of 5. After incubation with S-HyNic or S-4FB at room temperature for 2.5 hours, the modified antibody was desalted using a Zebra column and transferred to conjugation buffer. Equimolar amounts of S-HyNic and S-4FB-modified antibodies were then mixed together, and 10x TurboLink catalysis buffer was added to the mixture at 1 / 10th the volume. The protein mixture was then incubated overnight at 4°C. On day 2 of the conjugation protocol, samples were centrifuged at 5000 g for 5 minutes to remove aggregates.

[0163] Antibody conjugation was confirmed by electrophoresis (NuPAGE gel, Invitrogen). The conjugated product was then purified using a HiLoad 16 / 600 Superdex™ 200 pg column (GE Healthcare Life Sciences) on fast protein liquid chromatography (FPLC) against PBS. After purification using FPLC, the molecular weights of different fractions of chemically conjugated BEAT were confirmed by electrophoresis (NuPAGE gel, Invitrogen). In some cases, the purified chemically conjugated BEAT was further concentrated using Vivaspins (Sartorius Stedim).

[0164] E. Functional analysis of SEB Human or mouse T cells were co-cultured with APCs and SEB, and 24 to 72 hours after co-culture, cell proliferation, IFN-γ secretion, and TCR Vβ8 uptake in the spleen were measured. + T cell infiltration was assessed.

[0165] To determine whether SEB could enhance the efficacy of CAR T cell therapy by enhancing T cell proliferation, we tested three different mouse cancer models: E0771-Her2 breast cancer, 24JK-Her2 sarcoma, and MC38-Her2 carcinoma transplanted into Her2 transgenic mice. These immunocompetent Her2 transgenic mice express human Her2 in the normal breast and cerebrum under the control of the whey acidic protein (WAP) promoter, making them resistant to Her2+ cell lines (Piechocki et al., 2003, Journal of Immunology, 171:5787-94). Once Her2+ tumors were established, CAR T cells were adoptively transferred, and two doses of SEB were administered i.p. on the same day and 5 days later.

[0166] F. Functional analysis of BEAT To assess the effect of BEAT on cell proliferation, a chemically conjugated bispecific polypeptide was generated from an anti-CD40 / anti-myc antibody. The c-myc tag already present on the Her2-specific CAR described above served as the CAR-specific antigen.

[0167] T cells, BEAT, and APCs will be co-cultured, and multiple assays will be used to confirm functional changes on T cells and APCs: cell proliferation (tritiated thymidine incorporation, CFSE labeling, and cell counting), cytokine secretion (ELISA, cytokine bead array assay, and intracellular staining), and cell activation / differentiation status (markers including CD25, CD69, CD45RA, CD45RO, CD27, CD28, MHCII, CD80, CD86, and CCR7) will be measured after 24-72 hours of co-culture.

[0168] The cytotoxic potential of BEAT-stimulated T cells will be assessed using a series of assays, including chromium release assays to test for cytolysis, granzyme B and CD107a staining on T cells, Annexin V / 7AAD, and real-time apoptosis / necrosis analysis (RealTime-Glo Annexin V Apoptosis and Necrosis Assay Kit, Promega). Appropriate BEAT, T cell, and tumor cell controls will be used to determine specificity.

[0169] To determine the ability of BEAT-generated CAR T cells to respond to various solid tumor cell lines, we will use the Her2-positive human cell lines MDA-MB-231 breast cancer, PANC1 pancreatic cancer, and A549 lung cancer cell lines (or controls) as targets after transplantation in immunodeficient mice, or the E0771-Her2, MC38-Her2, and 24JK-Her2 murine tumor cell lines in Her2-transgenic mice. Once Her2+ tumors are established, CAR T cells will be adoptively transferred, and two doses of BEAT will be administered i.p. on the same day and 5 days later.

[0170] result Generation of BEAT using mammalian systems Mammalian systems can be used to produce BEAT in soluble form and have the advantage of proper post-translational modifications, but the yields are considerably lower (i.e., less than 10 cells / mL). 8 (Less than 1 nmole of scFv protein was produced per cell).

[0171] Using the Expi293 cell expression system, we successfully generated scFv against CD3 (CAR-specific; SEQ ID NO: 6; Figure 1). Full-length BEATs were generated against BEAT2 (MHCII / c-myc; SEQ ID NO: 18) and BEAT3 (CD40 / CD3; SEQ ID NO: 20) using both the lipofectamine (Figure 2) and jetPEI (Figure 3) systems. However, the jetPEI system allowed more efficient release of recombinant BEAT after cell homogenization (Figure 4).

[0172] SEB enhanced T cell proliferation and IFN-γ secretion in vitro SEB is a bispecific polypeptide that specifically binds to MHCII molecules in the peptide-binding groove on APCs and outside the TCR on T cells bearing TCR Vβ chains, particularly Vβ3, 7, 8, and 17 in mice. Simultaneous binding of SEB to MHCII and TCR stimulates a significant number of both CD4+ and CD8+ T cells, leading to their activation and proliferation. While conventional antigens presented by MHCII activate 0.0001-0.001% of T cells, bispecific polypeptides such as SEB stimulate 2-20% of all T cells (Fraser and Proft, 2008, Immunology Reviews, 225:226-43; Arcus et al., 2000, Journal of Molecular Biology, 299(1):157-68). Thus, SEB was used as a proof-of-concept molecule to develop strategies for activating and expanding CAR T cells in vivo; however, the approach and reagents described herein achieve this goal in a more controlled manner with minimal toxic effects.

[0173] After incubation with SEB, mouse and human T cells proliferated to a significantly greater extent (Fig. 5A) and secreted greater amounts of IFN-γ in the presence of SEB (Fig. 5B). When SEB was injected into mice bearing established E0771-Her2 breast cancer, the percentage of TCR-Vβ8+ (one of the TCRβ types responsible for SEB engagement) cells in the spleen was significantly higher (Fig. 5C) than in the untreated group (Fig. 5C).

[0174] Strikingly, tumor growth was significantly inhibited in all three mouse models after adoptive transfer of CAR T cells and two doses of SEB, with 30-50% of mice becoming tumor-free and achieving long-term survival (Figure 6). Tumors were inhibited to a significantly greater extent in mice receiving the combination of CAR T cells and SEB compared to mice receiving CAR T cells or SEB alone.

[0175] Tumor suppression was associated with a >10-fold increase in CAR T cell infiltration into the tumor site (Figure 7). The number of CAR T cells per dose that were of the correct Vβ type for SEB engagement was approximately 2 x 10 5 (10% of total transfected CAR T cells). The high degree of tumor inhibition without toxicity observed with such a low effective dose of CAR T cells in an immunocompetent model represents a significant and unexpected result compared to other known effective doses for CAR T cells, and suggests that even greater efficacy is possible when engagement of CAR T cells with APCs occurs in a non-MHC-restricted or non-Vβ-restricted manner.

[0176] CAR T cell activation and proliferation by BEAT Importantly, it was confirmed that binding of anti-myc to CAR T cells did not interfere with CAR-mediated T cell function (Figure 8). Furthermore, it was also demonstrated that chemically conjugated bispecific polypeptides greatly enhanced CAR T cell proliferation in vitro (Figure 9). In contrast, the combination of two single antibodies at the same molar concentration did not mediate CAR T cell proliferation. These data suggest that the bispecific polypeptides engage APCs together with CAR T cells, and that this engagement results in enhanced CAR T cell proliferation.

[0177] BEAT mediates CAR T cell activation and proliferation Three examples of BEAT were generated by chemically conjugating an antibody specific for the myc tag (present in the CAR) to antibodies specific for either CD40, PD-L2, or Clec9a expressed on various subsets of APCs. Mouse CAR T cells were incubated with mouse splenocytes (as the source of APCs) in the presence or absence of BEAT. T cells lacking CAR expression were used as a control. Incubation of CAR T cells with APCs in the presence of unconjugated antibody was also used as a control.

[0178] The results shown in Figure 10 demonstrate that in the presence of each BEAT, CAR T cells, but not control T cells, secrete IFN-γ (Figure 10A).

[0179] CAR T cells were loaded with the fluorescent dye CFSE and incubated with APCs in the presence or absence of each surrogate BEAT. The extent of proliferation is evident from the decrease in fluorescence intensity of transfected CAR T cells compared to control untransfected T cells (Figure 10B).

[0180] Taken together, these results demonstrate that BEAT can activate CAR T cells and induce their proliferation, and further indicate that various APC molecules and APC subsets can be used as targets for BEAT.

[0181] CD40-myc BEAT enables eradication of E0771-Her2 breast cancer in mice Her2-transgenic mice bearing established subcutaneous E0771-Her2 tumors were transfected with CAR T cells (2 × 10 6 , iv) and / or BEAT specific for myc-CD40 (36 μg, ip on day 0). Other groups of mice received unconjugated anti-myc and anti-CD40 antibodies or were left untreated (control).

[0182] The results in Figure 11 show tumor growth and mouse survival following administration of BEAT coupled with CAR T cells. The data demonstrate the ability of CD40-myc BEAT to enable CAR T cell-mediated eradication of established breast cancer tumors in immunocompetent mice expressing Her2 in the normal mammary gland and brain.

[0183] CD40-myc BEAT enables T cell accumulation within tumors Mice bearing subcutaneous E0771-Her2 tumors were treated with anti-HER2 CAR T cells (2 × 10 6 , iv) along with CD40-myc BEAT (36 μg, ip on day 0). Other mice received CAR T cells or BEAT alone, or were left untreated (control).

[0184] On day 7 after the start of treatment, tumors were harvested, dissociated, stained to detect CD8+ T cells, and analyzed using flow cytometry. Data represent three individual mice per group. Tumors were formalin-fixed, sectioned, and stained with DAPI (blue) and anti-CD8 (brown). Images are shown representing three sections from mice receiving the listed treatments.

[0185] The results shown in Figure 12 demonstrate the ability of BEAT to enable CAR T cells to accumulate within tumors to a much greater extent than in the absence of BEAT.

[0186] CAR T cells persist long-term in mice Spleens were harvested from naive or long-term (>200 days) surviving mice after eradication of E0771-Her2 tumors by CAR T cells + CD40-myc BEAT and stained with anti-myc and CD8 to detect CAR T cells. Data representative of 3 mice.

[0187] The data shown in Figure 13 demonstrate the ability of BEAT to enable long-term persistence of CAR T cells in mice, suggesting that the mice will be protected from tumor re-challenge.

[0188] Mice that reject Her2-positive tumors after CAR T cell plus BEAT therapy are completely resistant to rechallenge with Her2-positive tumors Long-term survivors of subcutaneous E0771-Her2 tumor eradication mediated by Her2-specific CAR T cells and CD40-myc BEAT were treated with the same Her2-positive tumor cells (5 × 10 5 The mice were re-administered subcutaneously on the opposite flank using the IVF-10 ... *** p<0.001, **** p<0.0001).

[0189] These data support the conclusion that surviving CAR T cells (transplanted) after CAR T+BEAT therapy can respond to subsequent recurrence of Her2-positive tumors.

[0190] Her2-negative tumor growth is inhibited in mice that reject Her2-positive tumors mediated by CAR T cells and BEAT therapy Mice that had previously rejected E0771-Her2 tumors after receiving Her2-specific CAR T cells and CD40-myc BEAT were treated with E0771 (5 × 10 cells) lacking Her2 expression. 5 The tumor growth rate (upper panel) and mouse survival rate (lower panel) are shown in Figure 15 (four mice in the re-administration group and five mice in the control group). ** p<0.01, *** p<0.001, **** p<0.0001).

[0191] These data suggest that CAR T cell + BEAT therapy induces epitope spreading, thereby inducing T cells with specificities other than Her2, which may participate in antitumor activity.

[0192] Anti-Her2 CAR T cells combined with CD40-myc-conjugated BEAT inhibit growth of MC38-Her2 colon cancer in mice Mice bearing established subcutaneous MC38-Her2 tumors were transfected with anti-Her2 CAR T cells (2 × 10 6 , iv) and CD40-myc BEAT (36 μg on day 0). Other groups of mice received CAR T cells or (listed) antibodies alone, or were left untreated (control). (6 mice per group, except for the BEAT+CAR group, which had 7 mice. ** p<0.01).

[0193] The results shown in Figure 16 indicate that tumor types other than breast cancer can be controlled using CAR T cells plus BEAT.

[0194] PD-L2-myc and Clec9a BEAT conjugates enable CAR T cell-mediated inhibition of tumor growth BEATs specific for APC-expressed molecules PD-L2 or Clec9a were prepared by chemical conjugation of monoclonal antibodies to anti-myc antibodies. Mice bearing established subcutaneous E0771-Her2 tumors received the listed treatments or were left untreated (control) (3-6 mice per group). * p<0.05, ** p<0.01).

[0195] The results shown in Figure 17 demonstrate that BEATs specific for several different molecules found on various APC subsets can enhance CAR T cell responses to tumors in mice.

[0196] Recombinant human CD40-myc BEAT mediates human CAR T cell expansion CAR T cells were generated from human peripheral blood using retroviral transduction of Her2-specific CAR. CAR T cells (2 × 10 5 T cells (3 μg / ml) were labeled with CFSE and incubated for 5 days with or without chemically conjugated or recombinant CD40-myc BEAT (3 μg / ml conjugated, 0.5 μg / ml recombinant) in the presence of 5 Gy-irradiated CTV-labeled human blood leukocytes as a source of APCs. T cells transfected with an empty vector served as a control. Cells were analyzed using flow cytometry.

[0197] The results shown in Figure 18 demonstrate that BEAT linked to anti-myc with anti-human CD40 can mediate human CAR T cell proliferation. The results further demonstrate that recombinant BEAT is as effective as chemically conjugated BEAT in mediating CAR T cell proliferation.

[0198] Recombinant human CD40-myc BEAT enhances IFN-γ secretion from CAR T cells Human CAR T cells were generated from peripheral blood using a retroviral vector encoding an anti-Her2 CAR. CAR T cells were incubated with human blood leukocytes as a source of APCs for 5 days in the presence or absence of chemically or recombinantly prepared CD40-myc BEAT. Anti-CD3 was included in some culture wells as a positive control. Supernatants were collected and analyzed for IFN-γ using ELISA.

[0199] As shown in Figure 19, recombinant BEAT can activate human CAR T cells to secrete IFN-γ.

[0200] Human CAR T cells combined with conjugated human BEAT inhibit tumor growth in mice Nod-SCID-γ (NSG) mice bearing established subcutaneous MDA-MB-231 human mammary tumors received CAR T cells with or without conjugated human CD40-myc BEAT (day 0, 36 μg, i.p.). Human blood-derived leukocytes (1 × 10) were administered to serve as a source of APCs. 6 ) was also delivered intravenously. One group of mice was left untreated as a control (5 mice per group, * p<0.05, ** p<0.01, *** p<0.001, Student's t-test).

[0201] The data shown in Figure 20 demonstrate the ability to enhance the efficacy of human CAR T cells against established human cancers in mice.

[0202] BEATs specific for a range of antigens located at various locations within the CAR can induce responses from CAR T cells The ability of BEAT to be specific for a range of antigens within a CAR was assessed by generating CARs with antigens (i.e., affinity tags) incorporated at different locations within the CAR. Specifically, a carcinoembryonic antigen (CEA)-specific CAR was prepared by incorporating a centrally located myc antigen within the CAR, and a Her2-FLAG CAR was prepared by incorporating a FLAG antigen within the amino terminus of the CAR.

[0203] 5 x 10 carrying CEA-myc CAR or Her2-FLAG CAR 5 5 × 10 mouse CAR T cells 5 CD40-positive mouse splenocytes (as a source of APCs) were incubated with or without BEAT specific for the APC-expressed molecule CD40 (BEAT1; described elsewhere herein) prepared by chemical conjugation of a monoclonal antibody to an anti-myc antibody or BEAT specific for the APC-expressed molecule CD40 (BEAT5) prepared by chemical conjugation of a monoclonal antibody to an anti-FLAG antibody. Untransfected splenocytes were used as a control. Incubation of CAR T cells with APCs in the presence of unconjugated antibodies was also used as a control. IFN-γ secretion was determined by ELISA after 72 hours of coculture.

[0204] The results shown in Figure 21 demonstrate that CEA-myc CAR T cells secreted IFN-γ in the presence of BEAT1 compared to the control. Similarly, HER2-FLAG CAR T cells secreted IFN-γ in the presence of BEAT5 compared to the control.

[0205] These results demonstrate that BEAT has broad applications in improving the efficacy of CAR T-cell therapy. First, BEAT has been shown to induce responses from CAR T cells, where the CAR T cells are directed against various tumor-associated antigens.

[0206] Second, BEAT has been shown to induce responses from T cells when CAR T cells are used in the context of cancers occurring in a variety of tissue types.

[0207] The broad utility of BEATs is further evidenced by the fact that their design can be modified to increase the efficacy of a variety of different CAR structures in the context of CAR T therapy. For example, the results presented herein demonstrate that BEATs can be designed to bind to a variety of antigens on a CAR and retain the ability to induce a response from CAR T cells. Thus, a range of antigens within a CAR can be used as targets for BEATs.

[0208] Importantly, it has also been demonstrated that the location of the antigen on the CAR can be varied without significantly affecting the efficacy of BEAT. More specifically, both a centrally located myc tag (i.e., Her2-myc and CEA-myc CARs) and an N-terminally located FLAG epitope (i.e., Her2-FLAG CAR) have been shown to be useful binding targets for BEAT. These data further demonstrate that antigens located at various positions within the CAR are effective for inducing CAR T cell responses.

[0209] conclusion The results presented herein demonstrate that BEATs can be designed and tailored for use in conjunction with a wide range of different CAR structures to increase the efficacy of CAR T therapy.

[0210] The bispecific polypeptides (i.e., BEATs) generated in this study have proven effective in activating and expanding CAR T cells through APC engagement. Furthermore, BEATs have proven effective in targeting various tumor antigens and / or a variety of different antigens at different locations within the CAR. This approach is particularly advantageous compared to other methods of supporting T cell therapy because there are no restrictions regarding MHC haplotypes.

[0211] The pharmacokinetics of BEAT advantageously induces "on-demand" activation and proliferation, thereby allowing for greater control over CAR T cells by balancing the demands of anti-tumor efficacy with controlled cytotoxicity to normal tissues, which has not been achieved using other methods of CAR T cell support, such as vaccines.

Claims

1. 1. A bispecific polypeptide comprising a first binding domain and a second binding domain, wherein the first binding domain is an antibody or antibody fragment that specifically binds to an antigen expressed on an antigen-presenting cell (APC), and the second binding domain is an antibody or antibody fragment that specifically binds to an antigen on an immune cell that expresses a chimeric antigen receptor (CAR).

2. 2. The bispecific polypeptide of claim 1, wherein the second binding domain specifically binds to an antigen on the CAR expressed by the immune cell.

3. 3. The bispecific polypeptide of claim 1, wherein the APC is a professional APC selected from the group consisting of dendritic cells, macrophages and B cells, preferably the APC is a dendritic cell.

4. 4. The bispecific polypeptide of claim 1 , wherein the first binding domain comprises a single-chain variable fragment (scFv).

5. 5. The bispecific polypeptide of claim 1, wherein the second binding domain comprises an scFv.

6. 6. The bispecific polypeptide of claim 1 , wherein the first binding domain specifically binds to an antigen expressed on the APC selected from the group consisting of MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, and CD83.

7. The bispecific polypeptide of claim 6 , wherein the APC is not a tumor cell or a cancer cell.

8. 8. The bispecific polypeptide of claim 6 or 7, wherein the antigen expressed on the APC is not a tumor-associated antigen.

9. 9. The bispecific polypeptide of claim 1, wherein the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte, a tumor infiltrating lymphocyte (TIL), and a regulatory T cell.

10. The bispecific polypeptide of claim 9 , wherein the immune cell is a T cell.

11. 11. The bispecific polypeptide of claim 1 , wherein the second binding domain specifically binds to an affinity tag on the CAR.

12. 11. The bispecific polypeptide of claim 1, wherein the second binding domain specifically binds to an affinity tag on an immune cell.

13. 11. The bispecific polypeptide of claim 1, wherein the second binding domain specifically binds to CD3.

14. 13. The bispecific polypeptide of any one of claims 2 to 12, wherein the second binding domain specifically binds to an affinity tag selected from a c-myc tag, a FLAG tag, an HA tag, a His tag, an S tag, an SBP tag, a Strep tag, an eXACT tag, a GST tag, an MBP tag, or a GFP tag.

15. The bispecific polypeptide of claim 14, wherein the affinity tag is a c-myc tag.

16. 16. The bispecific polypeptide of any one of claims 1 to 15, wherein the first binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or 4, or an amino acid sequence having at least 70% identity thereto.

17. 14. The bispecific polypeptide of claim 13, wherein the second binding domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 70% identity thereto.

18. 16. The bispecific polypeptide of any one of claims 1 to 11, 14 and 15, wherein the second binding domain comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 70% identity thereto.

19. 19. The bispecific polypeptide of any one of claims 1 to 18, further comprising a flexible linker connecting the first and second binding domains, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 12 or 14, or an amino acid sequence having at least 70% identity thereto.

20. 19. The bispecific polypeptide of claim 1, wherein the first and second binding domains are chemically conjugated.

21. 16. The bispecific polypeptide of any one of claims 1 to 11, 14 and 15, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 18, or an amino acid sequence having at least 70% identity thereto.

22. 14. The bispecific polypeptide of any one of claims 1, 3 to 10 and 13, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 22, or an amino acid sequence having at least 70% identity thereto.

23. 23. The bispecific polypeptide of any one of claims 1 to 22, characterized by a plasma half-life of about 5 hours to about 15 hours.

24. 24. The bispecific polypeptide of claim 23, wherein the plasma half-life is from about 10 hours to about 12 hours.

25. 25. A nucleic acid sequence encoding the bispecific polypeptide of any one of claims 1 to 24, or a polynucleotide sequence having at least 70% identity thereto.

26. 26. The nucleic acid sequence of claim 25, comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15, 17, 19 or 21, or a polynucleotide sequence having at least 70% identity thereto.

27. 27. A vector comprising the nucleic acid sequence of claim 25 or 26 operably linked to a regulatory sequence.

28. 28. The vector of claim 27, comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26.

29. A cell comprising the vector of claim 27 or 28.

30. 30. A method for producing a bispecific polypeptide, comprising: (i) culturing the cells of claim 29 in a culture medium and under conditions suitable for expression of said bispecific polypeptide; and (ii) isolating said bispecific polypeptide from said cells or said culture medium.

31. 25. A pharmaceutical composition comprising the bispecific polypeptide of any one of claims 1 to 24 and a pharmaceutically acceptable carrier.

32. 32. A method for treating cancer, comprising the step of co-administering to a subject in need thereof therapeutically effective amounts of: (i) immune cells expressing a CAR; and (ii) the bispecific polypeptide of any one of claims 1 to 24 or the pharmaceutical composition of claim 31, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on endogenous APCs of the subject and to an antigen on the CAR expressed by the immune cells, to stimulate the activation and proliferation of the immune cells in vivo to treat cancer.

33. 32. A method for stimulating the activation and proliferation of immune cells in vivo, comprising the step of co-administering to a subject effective amounts of: (i) immune cells expressing a CAR; and (ii) the bispecific polypeptide of any one of claims 1 to 24 or the pharmaceutical composition of claim 31, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on an endogenous APC of the subject and to an antigen on the CAR expressed by the immune cells, thereby stimulating the activation and proliferation of the immune cells in vivo.

34. 24. A method for stimulating the activation and proliferation of immune cells in vitro, comprising the step of culturing isolated immune cells expressing a CAR in a culture medium comprising: (i) an APC or an APC mimetic, and (ii) the bispecific polypeptide of any one of claims 1 to 23, wherein the bispecific polypeptide simultaneously binds to an antigen expressed on the APC or APC mimetic and to an antigen on the CAR expressed on the immune cell, thereby stimulating the activation and proliferation of the immune cell in vitro.

35. The method according to any one of claims 32 to 34, wherein the immune cells are selected from the group consisting of T cells, NK cells, cytotoxic T lymphocytes, TILs and regulatory T cells.

36. 36. The method of claim 35, wherein the immune cell is a T cell.

37. The method of any one of claims 32 to 36, wherein the immune cells are derived from autologous cells.

38. The method of any one of claims 32 to 36, wherein the immune cells are derived from heterologous syngeneic cells.

39. 39. The method of any one of claims 32 to 38, wherein said bispecific polypeptide stimulates said activation and proliferation of said immune cells in an MHC-halotype-independent manner.

40. 40. The method of any one of claims 32 to 39, wherein the bispecific polypeptide specifically binds to the subject's endogenous professional APC, optionally selected from the group consisting of dendritic cells, macrophages, and B cells.

41. 41. The method of claim 40, wherein the bispecific polypeptide does not bind to an antigen on a tumor cell or cancer cell.

42. 42. The method of any one of claims 32 to 41, wherein the antigen bound by the bispecific polypeptide is not a tumor-associated antigen.

43. 32. Use of the bispecific polypeptide of any one of claims 1 to 24 or the pharmaceutical composition of claim 31 in the manufacture of a medicament for treating cancer.

44. 32. The bispecific polypeptide of any one of claims 1 to 24 or the pharmaceutical composition of claim 31 for use in the treatment of cancer.

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