How to rescue exhausted immune cells

Abstract

The present invention is directed to compositions and methods for preventing or rescuing exhaustion of immune effector cells, particularly CAR T cells. The compositions and methods comprise a bispecific polypeptide having a first antigen binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (e.g., CD40) and a second antigen binding protein that specifically binds to an antigen on an immune effector cell (e.g., a FLAG tag on a CAR T cell).

Description

[Technical Field]

[0001] The present disclosure relates generally to methods for reducing, preventing, or rescuing exhausted immune cells that express engineered receptors (e.g., chimeric antigen receptors or modified TCRs).

[0002] Related Applications This application claims priority from Australian provisional application AU2022902711, the entire disclosure of which is incorporated herein. [Background technology]

[0003] Immune cell exhaustion is a state of acquired immune cell dysfunction. Specifically, T cell exhaustion, a state of acquired T cell dysfunction, is a hallmark of cancer and chronic viral infections. Recently, treatments that reverse T cell exhaustion in cancer have proven remarkably effective.

[0004] The use of engineered immune cells expressing genetically engineered antigen receptors (e.g., chimeric antigen receptor (CAR) T cells) has also proven effective for the treatment of hematological malignancies. However, the occurrence of exhaustion in CAR T cells remains a major barrier to the widespread use of this technology, especially in the treatment of solid tumors.

[0005] T cell exhaustion (including CAR T exhaustion) is characterized by a progressive decline in effector function, usually manifested as a loss of T cell polyfunctionality. Exhausted T cells typically exhibit increased expression of inhibitory receptors (e.g., PD-1, LAG-3, and PD-L1), reduced production of effector cytokines, slowed proliferation rate, and reduced target cell killing activity. Regenerating exhausted T cells can reinvigorate immunity.

[0006] Current strategies for modulating T cell exhaustion rely on directly regulating the expression of effector gene expression products, such as immune checkpoints. Such modulation often results in undesirable side effects, as physiological levels of such effector gene expression products are often necessary for normal T cell function. Furthermore, such strategies are vulnerable to drug resistance and can lead to immunopathology.

[0007] Thus, there is a need to develop new compositions and methods for preventing or rescuing immune cell exhaustion, particularly T cell exhaustion.

[0008] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or reasonably expected to be incorporated into other pieces of prior art by those skilled in the art. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Patent Application Publication No. WO2014 / 190273 [Patent Document 2] International Patent Publication No. WO2013 / 188864 [Patent Document 3] International Patent Publication No. WO97 / 34636 [Non-patent literature]

[0010] [Non-Patent Document 1] J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984) [Non-patent document 2] J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) [Non-patent document 3] T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991) [Non-patent document 4] DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996) [Non-patent document 5] F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions) [Non-patent document 6] Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988) [Non-Patent Document 7] J.E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all current revisions) [Non-patent document 8] Ward et al., 1989, Nature, 341:544-6 [Non-Patent Document 9] Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 [Non-Patent Document 10] Bork, J Mol. Biol. 242, 309-320, 1994

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[0011] The present invention provides a method of rescuing immune effector cell activity or reversing immune effector cell exhaustion, comprising the step of exposing immune effector cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby rescuing immune effector cell activity or reversing immune effector cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell; - optionally, the immune effector cells express a heterologous receptor comprising an antigen binding protein and a signaling protein; A method is provided.

[0012] The present invention also provides a method for increasing immune effector cell activity or cytotoxicity of exhausted immune effector cells, comprising exposing a plurality of immune effector cells that exhibit one or more symptoms of an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby increasing immune cell effector activity or cytotoxicity; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell; - optionally, the immune effector cells express a heterologous receptor comprising an antigen binding protein and a signaling protein; A method is provided.

[0013] The present invention also provides a method for preventing, inhibiting, reducing or delaying the onset of immune effector cell exhaustion, comprising exposing a plurality of immune effector cells that have or are suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby preventing, inhibiting, reducing or delaying the onset of immune effector cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell; - optionally, the immune effector cells express a heterologous receptor comprising an antigen binding protein and a signaling protein; A method is provided.

[0014] The present invention also provides a method for treating a disease or condition characterized by immune effector cell exhaustion, comprising the step of administering to a subject in need thereof an effective amount of a bispecific polypeptide, thereby treating the disease or condition characterized by immune effector cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell of the subject; A method is provided.

[0015] In any embodiment, the immune effector cell is selected from a T cell, an NK cell, an NKT cell, or a γδT cell. Preferably, the effector immune cell is a T cell.

[0016] Preferably, the heterologous receptor expressed by the immune effector cell is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) that binds to a target antigen (e.g., a tumor-associated antigen). It will therefore be appreciated that in preferred embodiments, the immune effector cell is a recombinant immune effector cell that expresses a CAR or modified TCR, or an immune effector cell derived therefrom.

[0017] Thus, in a preferred embodiment, the immune effector cells are T cells expressing a CAR (CAR T cells). Optionally, the immune effector cells are CAR NK cells or CAR NKT cells.

[0018] In a particularly preferred embodiment, a method for rescuing or reversing CAR T cell exhaustion comprises the step of optionally exposing a plurality of CAR T cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby rescuing or reversing CAR T cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on a CAR T cell; A method is provided.

[0019] As used herein, rescuing or reversing immune cell effector exhaustion includes reversing at least one marker or symptom of an exhausted phenotype in immune effector cells.

[0020] Further provided is a method for increasing CAR T cell activity or cytotoxicity, preferably comprising exposing a plurality of CAR T cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby increasing CAR T cell activity or cytotoxicity; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on a CAR T cell; A method is provided.

[0021] Further provided is a method for preventing, inhibiting, reducing the progression of or delaying the onset of CAR T cell exhaustion, preferably comprising exposing a plurality of CAR T cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby preventing, inhibiting, reducing or delaying the onset of CAR T exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on a CAR T cell; A method is provided.

[0022] In either embodiment, the step of exposing immune effector cells (preferably CAR T cells) to the bispecific polypeptide comprises contacting a population of immune effector cells (e.g., CAR T cells) obtained from the subject with an effective amount of the bispecific polypeptide ex vivo. Optionally, the method further comprises administering to the subject the immune effector cells that have been contacted with the bispecific polypeptide.

[0023] In either embodiment, the step of exposing immune cells (e.g., CAR T cells) to an effective amount of the bispecific polypeptide is carried out in vivo. Thus, in such embodiments, the method comprises administering the bispecific polypeptide to a subject who has undergone or is undergoing treatment with immune effector cells (e.g., CAR T cells), such that the present invention provides a method for producing, in a subject: - rescue immune effector cell (e.g., CAR T cell) activity; - Reverse immune effector cell (e.g., CAR T cell) exhaustion, - increase immune effector cell (e.g., CAR T cell) activity or cytotoxicity, or - Prevent, inhibit, reduce, or delay the development of immune effector cell (e.g., CAR T cell) exhaustion Provided are methods, the methods comprising the step of administering to a subject a bispecific polypeptide, wherein the bispecific polypeptide comprises a first antigen binding protein that specifically binds to an antigen expressed on an antigen presenting cell (APC), preferably a professional APC, and a second binding antigen binding protein that specifically binds to an antigen on an immune (e.g., CAR T) cell.

[0024] In preferred embodiments, the bispecific polypeptide is administered to a subject after the subject has received treatment with CAR T cells (or other immune effector cell treatment). In such embodiments, preferably the bispecific polypeptide is administered to the subject at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or at least 6 weeks or longer after administration of the CAR T cells (or other immune effector cell treatment) to the subject.

[0025] In either embodiment, at the time of administration of the bispecific polypeptide to a subject, one or more exhausted phenotypes of immune effector cells or markers or parameters indicative thereof are detectable or measurable in the subject or in a biological sample obtained from the subject.

[0026] In either embodiment, at the time of administration of the bispecific polypeptide to the subject, one or more exhausted immune effector cell phenotypes or markers or parameters indicative thereof have been detected or measured in the subject or in a biological sample obtained from the subject.

[0027] Optionally, at least or about 10%, at least 20%, at least 30%, at least 40%, or at least 50% or about 50% of the total immune effector cells (preferably CAR T cells) in a biological sample obtained from the subject have an exhausted phenotype or at least one sign or marker of an exhausted phenotype.

[0028] In either embodiment, the methods of the invention further comprise determining the risk or likelihood that the subject has immune effector cells (e.g., CAR T cells) with an exhausted phenotype.

[0029] As such, the present invention also provides a method of treatment comprising: - providing or identifying a subject who has undergone treatment with CAR T cells; - determining that the subject is likely to have or is at risk of having CAR T cells with an exhausted phenotype; - administering the bispecific polypeptide to the subject. Including, - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on a CAR T cell; A method is provided.

[0030] Determining whether a subject has or is at risk of having immune effector cells (e.g., CAR T cells) with an exhausted phenotype can include determining the expression of one or more genes associated with immune cell exhaustion in a sample of immune effector cells obtained from the subject.

[0031] In some embodiments, the exhaustion phenotype may comprise an increase in the level or degree of surface expression of markers on immune cells, or the percentage of immune cells exhibiting surface expression, of one or more exhaustion markers compared to a reference population of immune effector cells.

[0032] Examples of such genes or markers are further described herein and are known to those of skill in the art.

[0033] In any embodiment, the exhaustion phenotype comprises, for a T cell or population of T cells, an increase in the level or degree of surface expression on T cell(s), or the percentage of said population of T cells exhibiting surface expression, of one or more exhaustion markers, optionally 2, 3, 4, 5 or 6 exhaustion markers, compared to a reference T cell population under the same conditions, or a decrease in the level or degree of activity exhibited by said T cell or population of T cells when exposed to an antigen or antigen receptor-specific substance, compared to a reference T cell population under the same conditions.

[0034] In some embodiments, the reference immune effector cell population can be a population of immune effector cells known to have a non-exhausted phenotype, a population of naive T cells, a population of central memory T cells, or a population of stem central memory T cells. Optionally, the cells are from the same subject or of the same species as the subject from which the immune effector cells with the exhausted phenotype are derived.

[0035] In some embodiments, the reference immune effector cell population comprises (a) bulk immune effector cells isolated from the blood of the subject from which the immune effector cells with the exhausted phenotype are derived, where optionally the bulk immune effector cells are a subject-matched population that do not express a heterologous receptor (e.g., a CAR), and / or (b) obtained from the subject from which the immune effector cells with the exhausted phenotype are derived prior to receiving the dose of immune effector cells expressing a heterologous receptor (e.g., a CAR). In other embodiments, the reference immune effector cell population is a composition comprising an immune effector cell therapy sample or a pharmaceutical composition comprising immune effector cells expressing a recombinant receptor, prior to its administration to the subject, where optionally the composition is a cryopreserved sample.

[0036] The sample can be any biological sample containing immune cells, e.g., biological fluid, tissue, or tumor. Preferably, the biological sample is whole blood or a sample of immune cells derived therefrom (e.g., a sample of peripheral blood mononuclear cells, PBMCs).

[0037] The present invention also provides a method for producing a method for treating a subject comprising: - rescue or reverse immune effector cell (e.g., CAR T cell) exhaustion; - increase immune effector cell (e.g., CAR T cell) activity or cytotoxicity, or - Prevent, inhibit, reduce, or delay the development of immune effector cell (e.g., CAR T cell) exhaustion The present invention provides the use of a bispecific polypeptide in the manufacture of a medicament for treating a subject, preferably wherein the subject has undergone or is undergoing treatment with engineered immune effector cells (e.g., CAR T cells).

[0038] The present invention also provides - rescuing or reversing immune effector cell (e.g., CAR T cell) exhaustion; - increasing immune effector cell (e.g., CAR T cell) activity or cytotoxicity, or - Preventing, inhibiting, reducing, or delaying the development of immune effector cell (e.g., CAR T cell) exhaustion The present invention provides a bispecific polypeptide or a pharmaceutical composition comprising the same for use in

[0039] In any embodiment, the methods of the invention are for rescuing immune effector cell exhaustion in the context of any disease or condition selected from cancer, chronic infection (e.g., chronic bacterial or chronic viral infection), chronic inflammation, or autoimmunity. Thus, in certain embodiments, the methods of the invention include methods for treating a disease or condition, e.g., cancer, chronic infection, chronic inflammation, or autoimmunity, comprising administering to a subject in need thereof a bispecific polypeptide as described herein.

[0040] As used herein, a bispecific polypeptide comprising a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on a CAR T cell may also be referred to as a "BEAT."

[0041] Preferably, the professional APCs are endogenous APCs of the subject in need of treatment. Preferably, the professional APCs are selected from dendritic cells, macrophages, and B cells, more preferably dendritic cells (DCs). Thus, in a preferred embodiment, the first antigen-binding protein of the bispecific polypeptide can bind to any antigen of a professional APC, for example, any selected from MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, CD206, and CD83.

[0042] Preferably, the second antigen binding protein of the bispecific polypeptide specifically binds to an antigen on the CAR portion of the CAR T cell. The second antigen binding protein of the bispecific polypeptide may specifically bind to the antigen binding protein of the CAR, the hinge region of the CAR, a tag on the CAR (e.g., Myc, Flag, His, HA, SBP, GST, MBP, GFP, S, Strep, eXact or other tag), or any other region of the extracellular portion of the CAR.

[0043] Alternatively, the second antigen binding protein of the bispecific polypeptide may specifically bind to an antigen on an immune effector cell (optionally, the immune effector cell expresses a heterologous receptor, e.g., a CAR). In such embodiments, the second antigen binding protein of the bispecific polypeptide may specifically bind to an antigen on the surface of an immune effector cell (e.g., a T cell), such as CD3, CD4, CD8, CD25, CD127, CD196 (CCR6), CD27, CD28, CD45RA, CD45RO, CD62L, CD197, and HLA-DR. In further embodiments, the immune effector cell may be engineered to express a heterologous antigen or tag in addition to the CAR receptor, and the second antigen binding protein of the bispecific polypeptide may bind to the heterologous antigen or tag.

[0044] In a preferred embodiment, the bispecific polypeptide is in the form of a fusion protein, optionally wherein the first and second antigen-binding proteins are linked directly or via a hinge or linker region. Suitable linkers are further described herein.

[0045] The first and second antigen-binding proteins of the bispecific polypeptide are preferably in the form of an antibody or antigen-binding fragment thereof. In either embodiment, the first and / or second antigen-binding protein is in the form of an antibody, for example, immunoglobulin G (IgG1, IgG2, IgG3, or IgG4). Optionally, the first antigen-binding protein is in the form of an antibody, for example, immunoglobulin G (IgG1, IgG2, IgG3, or IgG4), and the second antigen-binding protein is in the form of an antibody fragment (e.g., scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2), and the second antigen-binding protein is linked to the first antigen-binding protein via the C- or N-terminus or an internal region of the first antigen-binding protein, preferably via the C-terminus of the heavy chain of the first antigen-binding protein. Optionally, the second antigen binding protein is in the form of an antibody, for example immunoglobulin G (IgG1, IgG2, IgG3 or IgG4), and the first antigen binding protein is in the form of an antibody fragment (e.g. scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2), and the first antigen binding protein is linked to the second antigen binding protein via the C- or N-terminus or an internal region of the second antigen binding protein, preferably the second antigen binding protein is linked to the second antigen binding protein via the C-terminus of the heavy chain of the second antigen binding protein.

[0046] In either embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD40 and a second antigen binding protein for binding to a tag on the CAR of the CAR T cell.

[0047] In either embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD206 and a second antigen binding protein for binding to a tag on the CAR of the CAR T-cell tag.

[0048] In either embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD40 and a second antigen binding protein for binding to the FLAG tag.

[0049] Preferably, the antigen binding protein for binding to CD40 is an activator of CD40.

[0050] In either embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD206 and a second antigen binding protein for binding to the FLAG tag.

[0051] In either embodiment the bispecific polypeptide comprises a first antigen binding protein for binding to CD40, wherein the first antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, The sequences of any framework regions and / or complementarity determining regions are as described herein, preferably as set out in Table 1.

[0052] In either embodiment, the first antigen binding protein competitively inhibits the binding to CD40 of an antibody or antigen-binding fragment thereof comprising a VH comprising the sequence set forth in SEQ ID NO:1 and a VL comprising the sequence set forth in SEQ ID NO:2.

[0053] In either embodiment, the first antigen binding protein comprises antigen binding domains CDRH1, CDRH2 and / or CDRH3 having variable heavy domains as defined in SEQ ID NO:1.

[0054] In either embodiment, the first antigen binding protein comprises antigen binding domains CDRL1, CDRL2 and / or CDRL3 having a light chain variable domain as defined in SEQ ID NO:2.

[0055] In any embodiment of the first antigen binding protein, it comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined in SEQ ID NO:1 and a variable light chain as defined in SEQ ID NO:2.

[0056] Preferably, the first antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, The linker can be a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0057] Optionally, the first antigen binding protein comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 4, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 5; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 6, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 8; or (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO:5; and (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 6; a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 8; Includes:

[0058] In some embodiments, the first antigen binding protein comprises a heavy chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 1, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

[0059] In some embodiments, the first antigen binding protein comprises a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:2, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

[0060] In either embodiment, the second antigen binding protein is for binding to a FLAG tag, and the second antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, an antigen-binding domain comprising where: FR1, FR2, FR3, and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a, and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; The sequences of any framework regions and / or complementarity determining regions are as described herein, preferably as set out in Table 1.

[0061] In either embodiment, the second antigen binding protein is capable of specifically binding to a FLAG tag or a variant thereof (such as defined in SEQ ID NO: 28 or 30, or as otherwise defined herein). The antigen binding protein is preferably capable of specifically binding to a protein domain comprising multiple FLAG tag sequences (e.g., 2xFLAG, 3xFLAG, etc.).

[0062] In either embodiment, the second antigen binding protein is for specifically binding to a peptide tag containing, or consisting of, the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 28). In addition to these sequences, other amino acids may be present in the FLAG tag to which the second antigen binding protein is bound; preferably, the additional amino acids in the FLAG tag are hydrophilic amino acids, such as R (Arg), D (Asp), E (Glu) and K (Lys), and / or amino acids with aromatic side chains, such as Y (Tyr), F (Phe), H (His) and W (Trp).

[0063] In a preferred embodiment, the second antigen-binding protein is capable of specifically binding to a FLAG-tag containing, comprising, or consisting of the sequence GDYKDDDDKG (SEQ ID NO:29), DYKDDDDK (SEQ ID NO:30), MDYKDDDDK (SEQ ID NO:31), DFKDDDK (SEQ ID NO:32), DYKAFDNL (SEQ ID NO:33), DYKDHDG (SEQ ID NO:34), MDFKDDDDK (SEQ ID NO:35), MDYKAFDNL (SEQ ID NO:36), DYKDHDI (SEQ ID NO:37), DYKDH (SEQ ID NO:38), DYKDD (SEQ ID NO:39), DYKDHD (SEQ ID NO:40) and / or DYKDDD (SEQ ID NO:41). The most preferred sequence is DYKDDDDK (SEQ ID NO:30). The term FLAG-tag also encompasses FLAG-tags that have been modified by amino acid insertion, deletion, or substitution, for example those derived from the FLAG-tags described above, in particular tags having the sequence DYKDDDDK.

[0064] In either embodiment, the FLAG tag is present at the N-terminus, C-terminus, or within the protein to which the bispecific polypeptide is capable of binding, binds, or specifically binds (e.g., a CAR).

[0065] In either embodiment, the second antigen binding protein competitively inhibits binding to a FLAG tag of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 15 and a VL comprising the sequence set forth in SEQ ID NO: 16.

[0066] In either embodiment, the second antigen binding protein comprises an antigen binding domain CDRH1, CDRH2 and / or CDRH3 having a variable heavy chain as defined in SEQ ID NO:15.

[0067] In either embodiment, the second antigen binding protein comprises antigen binding domains CDRL1, CDRL2 and / or CDRL3 having a variable light chain as defined in SEQ ID NO:16.

[0068] In either embodiment, the second antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain defined in SEQ ID NO: 15 and a variable light chain defined in SEQ ID NO: 16.

[0069] In any embodiment, the second antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, The sequences of any of the framework regions and / or complementarity determining regions are as described herein, preferably as set forth in Table 1, and optionally the linker can be a chemical, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0070] In any embodiment, the second antigen-binding domain comprises: a complementarity determining region CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 23; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO:25; and a VH comprising a CDR3 comprising, or consisting of, an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO:25. a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO:26; a CDR1 comprising or consisting of an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO:27; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 22; The antigen-binding domain comprises:

[0071] In any embodiment, the second antigen binding protein is A VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; and VL comprising CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22. The antigen-binding domain comprises:

[0072] In some embodiments, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

[0073] In some embodiments, the second antigen binding protein comprises an antigen binding domain comprising a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

[0074] In any embodiment of any aspect of the invention, the bispecific polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 42 and 43.

[0075] As used herein, unless the context requires otherwise, the term "comprise" and variations of this term, such as "comprising," "comprises," and "comprised," are not intended to exclude further additives, components, integers, or steps.

[0076] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and referring to the accompanying drawings, in which: [Brief explanation of the drawings]

[0077] [Figure 1] Figure 1 is a schematic diagram of the experimental protocol for stimulating CAR T cells with either high-expressing mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs), and restimulating with either tumor cells or MoDCs. [Figure 2] Figure 2 shows CAR T cell activation and exhaustion 6 days after stimulation of CAR T cells with either highly expressing mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs), followed by restimulation with either tumor cells or MoDCs. [Figure 3] Figure 3 shows CAR T cell phenotype after stimulation of CAR T cells with either high-expressing mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs), followed by restimulation with either tumor cells or MoDCs. [Figure 4] Figure 4 is a schematic diagram of the experimental protocol for sorting pre-stimulated CAR T cells for further stimulation and proliferation and phenotypic assessment, and then re-culturing the CAR T cells with mesothelin-positive tumor cells, mesothelin-negative tumor cells, monocyte-derived DCs (MoDCs), or monocyte-derived DCs (MoDCs) in combination with BEAT. [Figure 5] Figure 5 shows the T cell phenotype of CAR T cells pre-treated with BEAT and MoDCs (A) or tumor (B) using either BEAT and MoDCs, negative tumor, or positive tumor restimulation. CAR T cells pre-treated with BEAT and MoDCs have a higher central memory (TCM) and stem cell memory (TSCM) phenotype than CAR T cells pre-treated with tumors, which have a higher effector memory phenotype (TEM). [Figure 6]Figure 6 shows T cell proliferation in the case of CAR T cells pre-treated with either tumor restimulation (A) or BEAT and MoDC restimulation (B). CAR T cells pre-treated with BEAT and MoDC have higher proliferation than CARs pre-treated with tumor. DETAILED DESCRIPTION OF THE INVENTION

[0078] Array Description

[0079] [Table 1A]

[0080] [Table 1B]

[0081] [Table 1C]

[0082] [Table 1D]

[0083] Detailed Description of the Embodiments It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent in the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0084] Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, a reference to a single step, composition, group of steps, or group of compositions should be taken to include one and more (i.e., one or more) of those steps, compositions, group of steps, or group of compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, a reference to "a" includes the singular as well as two or more, a reference to "an" includes the singular as well as two or more, a reference to "the" includes the singular as well as two or more, etc.

[0085] Those skilled in the art will recognize that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of said steps or features, or any two or more thereof.

[0086] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0087] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in 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, Vols. 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (editors), Current Protocols in It is described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all current editions).

[0088] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0089] The inventors have surprisingly shown that bispecific polypeptides described herein for binding to professional APCs and to immune effector cells (e.g., CAR T cells) in vivo rescue the exhausted phenotype and promote the activation and expansion of exhausted immune cells. Thus, the methods of the present invention relate, in part, to methods for improving the efficacy of immune cell therapy, e.g., CAR T cell therapy.

[0090] definition The term "protein" should be taken to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other by covalent or non-covalent bonds (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using suitable chemicals or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. A protein can include one or more unnatural amino acids.

[0091] The term "polypeptide" or "polypeptide chain" will be understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0092] Amino acids may be referred to herein by their commonly used full name (e.g., cysteine), by their commonly known three letter symbol (e.g., Cys), or by the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission (e.g., C). Nucleotides may likewise be referred to by their commonly accepted single-letter codes.

[0093] The term "antibody," as used herein, broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains. Also disclosed herein are antigen-binding fragments, mutants, variants, and derivatives thereof that retain the essential epitope-binding characteristics of an antibody molecule. Such mutants, variants, and derivatives will be known to those of skill in the art, and illustrative examples are described elsewhere herein.

[0094] An antibody heavy chain typically comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region typically comprises three domains, CH1, CH2, and CH3. A light chain typically comprises 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), interspersed with framework regions (FRs). Each VH and VL typically comprises 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.

[0095] The term "antigen-binding fragment" or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. Illustrative examples of antigen-binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a 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 comprising a single variable domain (Ward et al., 1989, Nature, 341:544-6); and (vi) an isolated CDR.

[0096] As used herein, "variable region" refers to the portion of the light and / or heavy chain of an antibody defined herein that is capable of specifically binding to an antigen, and includes the amino acid sequences of the complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally, FR4) together with three CDRs. H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0097] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be found in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991; Bork et al., J. Mol. Biol. 242, 309-320, 1994; Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; Martin ("enhanced Chothia"; Mol. Immunol. (2008) 45:3832-3839; or Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997; or Giudicelli et al., Nucleic Acids Res. 25: This can be further clarified by considerations in the IMGT system discussed in 1997, pp. 206-211.

[0098] "Framework regions" (FR) are variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively; FR H1 is the V H FR 1 corresponds to V, and FR H2 corresponds to V H FR 2 corresponds to V, FR H3 corresponds to V H FR 3 corresponds to V, FR H4 corresponds to V HSimilarly, the FRs of VL are referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, and FR L1 corresponds to FR 4 of V L FR 1 corresponds to V, and FR L2 corresponds to V L FR 2 corresponds to V, and FR L3 corresponds to V L FR 3 corresponds to V, FR L4 corresponds to V L Compatible with FR 4.

[0099] As used herein, the term "Fv" refers to a V, whether composed of multiple polypeptides or a single polypeptide. L and V H The term "antigen-binding domain" should be taken to mean any protein with which V associates to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. H and V L The V may be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the invention (as well as any protein of the invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term should be understood to encompass fragments derived directly from antibodies as well as proteins corresponding to such fragments produced using recombinant means. In some instances, the V H is the heavy chain constant domain (C H ) 1 and / or V L is the light chain constant domain (C L Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, an scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., a CH2 or CH3 domain, e.g., a minibody.

[0100] A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of a whole antibody with the enzyme papain, resulting in a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin followed by reduction, resulting in a molecule consisting of an intact light chain and a portion of the heavy chain containing the VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing two Fab fragments linked, for example, using a leucine zipper or CH3 domain. A "single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0101] The term "antigen," as used herein, refers to a molecule bound by an "antibody," "antibody fragment," or "bispecific polypeptide." 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."

[0102] As used herein, the term "bind" in reference to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction containing labeled "A" and the protein will reduce the amount of labeled "A" bound to the antibody.

[0103] As used herein, the terms "binds specifically" or "specifically binds" should be taken to mean that an antigen binding protein of the invention reacts with or associates with a particular antigen or cell expressing it more frequently, more rapidly, for a longer period of time, and / or with greater affinity than does an alternative antigen or cell. For example, the antigen binding protein binds to an antigen with substantially greater affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than it binds to other related molecules. In examples of the present invention, the antigen binding protein "specifically binds" to an antigen with at least 1.5-fold or 2-fold or more (e.g., 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold) affinity than it binds to any other antigen. Generally, although not necessarily, reference to binding means specific binding, and each term should be understood to provide clear support for the other term.

[0104] The terms "engineered cells" and "genetically modified cells" can be used interchangeably herein. These terms refer to the inclusion and / or expression of a foreign gene or nucleic acid sequence that, in turn, modifies the genotype or phenotype of the cell or its progeny. In particular, these terms refer to the fact that cells, preferentially immune cells, can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins not naturally expressed in these cells. For example, immune cells can be engineered to express artificial constructs, such as chimeric antigen receptors, on their cell surface. For example, a CAR sequence can be delivered into cells using adenovirus, adeno-associated virus (AAV)-based, retrovirus, or lentivirus vectors, or any other pseudotyped variants thereof, or any other gene delivery mechanism, such as electroporation or lipofection using CRISPR / Cas9, transposons (e.g., Sleeping Beauty), or variants thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0105] The term "chimeric antigen receptor" or "CAR," as used herein, 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 of skill in the art, and illustrative examples include Her2, CEA, FBP, CD19, and BCMA.

[0106] 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. When tumor-associated antigens are not expressed by non-tumor cells (i.e., unique to tumor cells), they may be referred to as "tumor-specific antigens." When tumor-associated antigens are not unique to tumor cells, they are also expressed on non-tumor cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors may occur under conditions that allow the immune system to respond to the antigen. Tumor-associated antigens may be antigens expressed on non-tumor cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at low levels on normal cells but are expressed at significantly higher levels on tumor cells. Those tumor-associated antigens that are 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.

[0107] The term "cancer," as used herein, means any condition associated with abnormal cell proliferation. Such conditions would be known to one 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, pancreatic cancer, and lung cancer.

[0108] The terms "treat," "treatment," and "treating," as used herein, refer to any and all uses of curing a condition or symptom, or otherwise preventing, hindering, slowing, inhibiting, or reversing in any way the onset or progression of cancer or other undesirable symptoms. Thus, terms like "treating" should be considered in the broadest possible context. For example, treatment does not necessarily imply that a subject is treated until complete recovery or cure. In conditions exhibiting or characterized by multiple symptoms, treatment does not necessarily treat, prevent, hinder, slow, inhibit, or ameliorate all of the symptoms, but may treat, prevent, hinder, slow, inhibit, or ameliorate one or more of the symptoms.

[0109] The term "immune cell" or "immune effector cell" refers to a cell that performs a specific effector function and can be part of either the adaptive (i.e., cellular or humoral) or innate immune system, e.g., alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic precursor cell, e.g., pluripotent stem cells and early precursor subsets that can mature or differentiate into somatic cells. The cells can be naturally occurring or artificial / genetically modified cells (e.g., iPSCs and other artificial cell types) that can be generated by cytokine exposure. The immune cells can also be artificial cell subsets, including induced pluripotent stem cells and cells matured therefrom. Preferred immune cells are cells with cytotoxic effector function, such as alpha-beta T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or gamma-delta T cells. "Effector function" refers to the specialized function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0110] Treatment method The present invention relates to methods for rescuing or reversing immune effector cell exhaustion, increasing immune effector cell activity or efficacy (particularly in the case of exhaustion), and preventing, inhibiting, reducing the progression of, or delaying the onset of, immune effector cell exhaustion.

[0111] As described elsewhere herein, the immune effector cells whose exhaustion is to be prevented or rescued can be any immune effector cell, illustrative examples of which include T cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, natural killer T cells (NKT cells), and gamma-delta cells (γδ cells). T cells can be cytotoxic T cells (CTLs; CD8+ T cells), CD4+ cos+ T cells, CD4- cos- T cells, or any other subset of T cells. Other illustrative examples of suitable T cells include T cells that express one or more of the following markers: CD3, CD4, CD8, CD27, CD2S, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR.

[0112] In a preferred embodiment, the immune cells express a heterologous chimeric antigen receptor (CAR) or a modified T cell receptor (TCR). Such receptors and their use in the treatment of various conditions and diseases, particularly cancer, are well known to those skilled in the art.

[0113] Sources of cells for use in accordance with the methods of the present invention are known to those of skill in the art, and illustrative examples include peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue obtained from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, the cells are derived from whole blood.

[0114] In one embodiment, the immune cells are derived from autologous cells. In another embodiment, the immune cells are derived from allogeneic cells.

[0115] The term "autologous" refers to any material originating from the same individual that is to be subsequently reintroduced into the individual.

[0116] The term "allogeneic" refers to material derived from a different individual of the same species as the individual to 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 aspects, allogeneic material from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0117] It will be appreciated that in a preferred embodiment of the invention, the method relates to rescuing exhausted CAR T cells that have been administered to a subject and require reactivation.

[0118] Administration of the CAR-expressing immune cells and the bispecific polypeptide or pharmaceutical composition described herein may be achieved by formulating the immune cells and the bispecific polypeptide or pharmaceutical composition in the same composition (e.g., for simultaneous co-administration), or they may be formulated as different compositions for sequential administration.

[0119] It will be appreciated that given the focus of the methods of the present invention on rescuing immune cell exhaustion, activating immune cells that exhibit an exhausted phenotype, or preventing or delaying immune cell exhaustion, sequential administration may be preferred.

[0120] "Sequential" administration means that there is an interval between the administration of the immune cells and the bispecific polypeptide or pharmaceutical composition comprising same. The interval between sequential administrations can be a few seconds, minutes, hours, or days. Preferably, there is at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more, between the first administration of the immune cells (e.g., CAR T cells) and the bispecific polypeptide.

[0121] In one embodiment, periodic readministration of the bispecific polypeptide may be required to achieve the desired therapeutic effect.

[0122] In some embodiments, administration of the bispecific polypeptide is carried out in a cycling regimen comprising administration of an effective amount of the bispecific polypeptide (i) daily for a period of one week or more, (ii) per day for not more than six days per week for a period of one week or more, (ii) per day for not more than five days per week for a period of one week or more, or per day for not more than four days per week for a period of one week or more. In certain embodiments, administration of the bispecific polypeptide is carried out in a cycling regimen comprising administration of an effective amount of the bispecific polypeptide per day for not more than five days per week for a period of one week or more.

[0123] In some embodiments of any of the methods provided herein, administration of the bispecific polypeptide is initiated at or after peak or maximum levels of T cell therapy cells are detectable in the subject's blood. In some embodiments, administration of the bispecific polypeptide is initiated about 14 to about 35 days after initiation of administration of T cell therapy. In certain embodiments, administration of the bispecific polypeptide is initiated about 21 to about 35 days after initiation of administration of T cell therapy. In other embodiments, administration of the bispecific polypeptide is initiated about 21 to about 28 days after initiation of administration of T cell therapy. In some embodiments, administration of the bispecific polypeptide is initiated at or about 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days after initiation of administration of T cell therapy. In certain embodiments, administration of the bispecific polypeptide is initiated 28 days or about 28 days after initiation of administration of the T cell therapy. In some embodiments of any of the methods provided herein, at the time of initiation of administration of the bispecific polypeptide, the subject does not exhibit severe toxicity following administration of the T cell therapy.

[0124] In some embodiments of any of the methods provided herein, administration of the bispecific polypeptide is initiated 28 days or about 28 days after initiation of administration of the T cell therapy. In some embodiments of any of the methods provided herein, at the time of initiation of administration of the bispecific polypeptide, the subject does not exhibit severe toxicity after administration of the T cell therapy. In some embodiments, the severe toxicity is severe cytokine release syndrome (CRS), optionally Grade 3 or higher, prolonged Grade 3 or higher, or Grade 4 or 5 CRS, and / or the severe toxicity is severe neurotoxicity, optionally Grade 3 or higher, prolonged Grade 3 or higher, or Grade 4 or 5 neurotoxicity.

[0125] In some embodiments of any of the methods provided herein, if the subject experiences toxicity, optionally hematological toxicity, after administration of the bispecific polypeptide, administration of the bispecific polypeptide is withheld and / or the cycling regimen is modified. In some embodiments, the toxicity is selected from severe neutropenia, optionally febrile neutropenia, and prolonged Grade 3 or higher neutropenia. In some embodiments, administration of the bispecific polypeptide is resumed after the subject no longer experiences toxicity. In some embodiments, the cycling regimen is modified after administration of the bispecific polypeptide is resumed. In some embodiments, the modified cycling regimen comprises administering a reduced amount of the bispecific polypeptide and / or reducing the frequency of administration of the bispecific polypeptide. In certain embodiments, the modified cycling regimen comprises administering a reduced amount of the bispecific polypeptide.

[0126] The methods of the present invention also contemplate contacting immune cells with bispecific polypeptides ex vivo. For example, in a scenario where a patient has undergone treatment with immune cells (e.g., CAR T cells) and the cells now exhibit an exhausted phenotype, the cells can be harvested from the patient and contacted ex vivo with the bispecific polypeptide to rescue the exhausted phenotype and reactivate the immune cells. In such embodiments, the immune cells can also be subjected to a combined treatment with the bispecific polypeptides described herein and another known immune cell activator. Examples of such immune cell activation include providing a primary stimulatory signal, for example, via the T cell TCR / CD3 complex or by stimulation of the CD2 surface protein, and providing a secondary costimulatory signal via an accessory molecule, e.g., CD28 or 4-1BBL. In addition to the primary stimulatory signal provided via the TCR / CD3 complex or by CD2, a second, costimulatory signal is required for the induction of 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), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICE, HVEM, lymphotoxin beta receptor, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind B7-H3.

[0127] The subject in need of treatment may be a human or a mammal of economic and / or social importance to humans, such as non-human carnivores (e.g., cats and dogs), Sus genus animals (e.g., pigs, boars, and wild boars), ruminants (e.g., cattle, cows, sheep, giraffes, deer, goats, bison, and camels), horses, and endangered, zoo-kept, poultry, more particularly, domesticated poultry, including avian species such as turkeys, chickens, ducks, geese, and guinea fowl, because of their economic importance to humans. The term "subject" does not denote a particular age. Thus, adult, juvenile, and neonatal subjects are intended to be included.

[0128] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to any subject to which the present disclosure may be applicable. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. The subject may be at risk of developing an onset disease.

[0129] A "therapeutically effective amount," as used herein, means that the amount of bispecific polypeptide when administered to a mammal, particularly a human, in need of such treatment is sufficient to rescue or prevent an exhaustion phenotype in immune effector cells (e.g., CAR T cells). The exact amount of bispecific polypeptide 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.

[0130] In any embodiment, the provided methods can enhance T cell therapy, e.g., CAR-T cell therapy, and in some aspects, improve the outcome of treatment. In some embodiments, the methods are particularly advantageous in subjects in which the cells of the T cell therapy show poor expansion, are exhausted, show reduced or decreased persistence in the subject, and / or in subjects with cancer that is resistant or refractory to other therapies, has an aggressive or high-risk cancer, or has a relatively low response rate, or is likely to show a low response rate, to CAR-T cell therapy administered without the bispecific polypeptide, compared to another type of cancer or compared to administration with a different CAR-T cell therapy.

[0131] In some embodiments, provided methods are used at a time when T cell therapy (e.g., CAR T cells) may or are likely to exhibit characteristics of exhaustion. In some embodiments, the exhaustion phenotype becomes evident after T cells that have reached peak expansion begin to decline in number in the subject's blood. In some embodiments, the methods of exposing or contacting T cells of a T cell therapy (CAR T cells) with a bispecific polypeptide described herein are performed at a time when the T cells exhibit increased hypofunction or exhaustion compared to a time immediately prior to exposing the T cells to an antigen (baseline) or a time when the cells have been exposed to an antigen but continue to proliferate and have not yet reached peak expansion. In some embodiments, the increased hypofunction or exhaustion can be determined by increased expression of exhaustion markers compared to a previous time point. In some embodiments, the increased hypofunction or exhaustion, e.g., increased expression of exhaustion markers, is at a time point after administration of T cell therapy (e.g., CAR T cells) to a subject with a disease or condition associated with the antigen targeted by the T cell therapy. T cells after administration to a subject, e.g., T cells in the peripheral blood, can be monitored for markers of T cell activation or exhaustion, e.g., PD-1, TIM-3, and LAG-3, or as further described herein.

[0132] Methods for determining the exhaustion phenotype of immune effector cells are known to those of skill in the art and include determining the expression of various genes by the immune cells or determining the presence or absence of particular cell surface markers. In other words, those of skill in the art will be familiar with exhaustion markers and methods for assessing / determining them in samples of immune cells.

[0133] In some embodiments of any of the methods provided herein, the exhaustion phenotype with respect to a T cell or population of T cells comprises an increase in the level or degree of surface expression on T cell(s), or the percentage of said population of T cells exhibiting surface expression, of one or more exhaustion markers, optionally 2, 3, 4, 5, or 6 exhaustion markers, compared to a reference T cell population under the same conditions, or a decrease in the level or degree of activity exhibited by said T cell or population of T cells upon exposure to an antigen or antigen receptor-specific substance, compared to a reference T cell population under the same conditions. In some embodiments, the increase in level, degree, or percentage is greater than or equal to 1.2-fold, 1.5-fold, 2.0-fold, 3-fold, 4-fold, 5-fold, 6-fold, 60-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. In other embodiments, the level, degree, or percentage decrease is greater than or equal to 1.2-fold, 1.5-fold, 2.0-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

[0134] In some embodiments of any of the methods provided herein, the reference T cell population is optionally a population of T cells known to have a non-exhausted phenotype from the same subject or the same species as the subject from which the T cell(s) with the exhausted phenotype are derived, a population of naive T cells, a population of central memory T cells, or a population of stem central memory T cells.

[0135] In some embodiments of any of the methods provided herein, the reference T cell population (a) comprises bulk T cells isolated from the blood of the subject from which the T cell(s) with the exhausted phenotype are derived, where optionally the bulk T cells are a subject-matched population that do not express the recombinant receptor, and / or (b) is obtained from the subject from which the T cell(s) with the exhausted phenotype are derived prior to receiving the dose of T cells expressing the recombinant receptor. In some embodiments, the reference T cell population is a composition or pharmaceutical composition comprising a T cell therapy sample comprising T cells expressing the recombinant receptor prior to its administration to the subject, where optionally the composition is a cryopreserved sample.

[0136] In some embodiments of any of the methods provided herein, the one or more exhaustion markers are inhibitory receptors, ie, selected from among PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, and TIGIT.

[0137] In some embodiments of any of the methods provided herein, the activity is one or more of proliferation, cytotoxicity, or production of one or a combination of proinflammatory cytokines, optionally wherein the one or a combination of cytokines is selected from IL-2, IFN-gamma, and TNF-alpha. Other markers of immune cell activation include CD25 and CD69. In some embodiments, the exposure to the antigen or antigen receptor-specific substance comprises incubation with an antigen or antigen receptor-specific substance, optionally a substance that binds to a recombinant receptor, wherein the antigen is optionally a target antigen. In some embodiments, the antigen or antigen receptor-specific substance comprises an antigen-expressing target cell, optionally a cell of the disease, disorder, or condition. In certain embodiments, the target antigen is a human antigen.

[0138] It is within the skill of the art to assess the effectiveness of the treatments described herein according to the methods of the present invention. In other words, one skilled in the art can easily determine whether immune cell exhaustion has been prevented, rescued, or by assessing one or more markers of exhaustion, or by assessing one or more markers of immune cell activation.

[0139] In some embodiments of any of the methods provided herein, administration of the bispecific polypeptide reverses the exhaustion phenotype of T cells expressing the recombinant receptor in the subject that have an exhaustion phenotype; prevents, inhibits, or delays the onset of the exhaustion phenotype of T cells expressing the recombinant receptor in the subject; or reduces the level or degree of the exhaustion phenotype of T cells expressing the recombinant receptor in the subject; or reduces the percentage, total number of T cells expressing the recombinant receptor in the subject.

[0140] In some embodiments of any of the methods provided herein, initiation of administration of the bispecific polypeptide is performed subsequent to administration of the T cell therapy, and following administration or initiation of the bispecific polypeptide, the subject exhibits reversal or rescue of antigen- or tumor-specific activity or function of T cells expressing the recombinant receptor in said subject, optionally said reversal, rescue and / or administration of said bispecific polypeptide at a time after T cells expressing the recombinant receptor exhibit an exhausted phenotype in the subject or in the subject's blood.

[0141] In some embodiments of any of the methods provided herein, administering the bispecific polypeptide comprises administration in an amount, frequency and / or duration effective to (a) achieve increased antigen-specific or antigen receptor-driven activity of naive or non-exhausted T cells in a subject, optionally comprising T cells that express the recombinant receptor, following exposure of the T cells to an antigen or to an antigen receptor-specific substance, compared to without said administration of the bispecific polypeptide; or (b) prevent, inhibit or delay the development of an exhausted phenotype in naive or non-exhausted T cells in a subject, optionally comprising T cells that express the recombinant receptor, following exposure of the T cells to an antigen or to an antigen receptor-specific substance, compared to without said administration of the bispecific polypeptide; or (c) reverse an exhausted phenotype in exhausted T cells in a subject, optionally comprising T cells that express the recombinant receptor, compared to without said administration of the bispecific polypeptide in said subject.

[0142] In some embodiments, administering the bispecific polypeptide comprises administration in an amount, frequency and / or duration effective to (i) achieve said increase in activity, and (ii) prevent, inhibit or delay said onset of said exhaustion phenotype and / or reverse said exhaustion phenotype. In some embodiments, T cells in the subject comprise T cells that express said recombinant receptor, and / or said antigen is a target antigen.

[0143] In some embodiments of any of the methods provided herein, the dose of engineered T cells for administration either simultaneously or sequentially with respect to the bispecific polypeptide is 1 x 10, inclusive of each other. 5 ~5×10 8 or approximately 1 x 10 5 ~5×10 8 Total CAR-expressing T cells, 1 x 10 6 ~2.5×10 8 Total CAR-expressing T cells, 5 x 10 6 ~1×10 8 Total CAR-expressing T cells, 1 x 10 7 ~2.5×10 8Total CAR-expressing T cells, 5 x 10 7 ~1×10 8 In some embodiments of any of the methods provided herein, the dose of engineered T cells comprises at least or at least about 1 x 10 total CAR-expressing T cells. 5 CAR-expressing cells, at least or at least about 2.5 x 10 5 CAR-expressing cells, at least or at least about 5 x 10 5 CAR-expressing cells, at least or at least about 1 x 10 6 CAR-expressing cells, at least or at least about 2.5 x 10 6 CAR-expressing cells, at least or at least about 5 x 10 6 CAR-expressing cells, at least or at least about 1 x 10 7 CAR-expressing cells, at least or at least about 2.5 x 10 7 CAR-expressing cells, at least or at least about 5 x 10 7 CAR-expressing cells, at least or at least about 1 x 10 8 CAR-expressing cells, at least or at least about 2.5 x 10 8 CAR-expressing cells or at least about or at least 5 x 10 8 In certain embodiments, the dose of engineered T cells comprises 5 x 10 CAR-expressing cells. 7 or approximately 5 x 10 7 In other embodiments, the dose of engineered T cells comprises 1 x 10 total CAR-expressing T cells. 8 or approximately 1 x 10 8 Contains CAR-expressing cells.

[0144] In some embodiments of any of the methods provided herein, the dose of cells is administered parenterally, optionally intravenously.

[0145] In some embodiments of any of the methods provided herein, the T cells are primary T cells obtained from a subject. In some embodiments, the T cells are autologous to the subject. In other embodiments, the T cells are allogeneic to the subject.

[0146] In some embodiments of any of the methods provided herein, the dose of engineered T cells comprises CD4+ T cells expressing a CAR and CD8+ T cells expressing a CAR, and administering the dose comprises administering a plurality of separate compositions, the plurality of separate compositions comprising a first composition comprising one of the CD4+ T cells and the CD8+ T cells, and a second composition comprising the other of the CD4+ T cells or the CD8+ T cells.

[0147] The dose of bispecific polypeptide used for direct administration to a subject or exposure to immune cells in vitro can be determined by one of skill in the art and typically varies depending 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 identified as being at high risk, at higher risk, or having developed recurrent disease may be prescribed a more aggressive treatment regimen compared to a subject considered to be at low or lower risk of developing recurrent disease. Similarly, a subject identified as having a more advanced stage of cancer, e.g., stage III or IV disease, may be prescribed a more aggressive treatment regimen compared to a subject with a less advanced stage of cancer.

[0148] Bispecific Polypeptides As used herein, the term "bispecific polypeptide" refers to a polypeptide that can specifically bind to two different target antigens simultaneously. Bispecific polypeptides for use in the methods described herein contain two structurally distinct antigen-binding domains or proteins (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 effector cell.

[0149] Preferably, the binding protein comprises an antigen binding protein for binding to a target antigen on professional APCs, more preferably endogenous professional APCs, of the subject in need of treatment. Examples of such antigens and such cells are further described herein.

[0150] Preferably, the immune effector cells are immune effector cells that express a heterologous receptor, e.g., a chimeric antigen receptor (CAR), comprising an antigen-binding domain and a signaling domain. Thus, in a preferred embodiment, the bispecific polypeptide comprises an antigen-binding domain for binding to an antigen on an immune cell expressing the CAR (e.g., a CAR T cell or a CAR NK cell).

[0151] It will be appreciated that bispecific polypeptides can be used to bind to a target antigen on an APC and to a target antigen on a heterologous receptor (e.g., a CAR) expressed by an immune cell.

[0152] The antigen-binding protein of a bispecific polypeptide for use in accordance with the present invention may comprise an antibody or antigen-binding fragment thereof. For example, the bispecific polypeptide may comprise the polypeptide sequence (i.e., domain) of one or more antibodies or antibody fragments. Examples of antibody fragments are described further herein.

[0153] In any embodiment, a bispecific polypeptide for use in the methods described herein may comprise a tandem single-chain variable fragment antibody (taFv) having a first scFv and a second scFv.

[0154] The bispecific polypeptides for use in the methods described herein may be variously referred to as "bispecific engagers of APCs and T cells" or "BEATs."

[0155] The term "antigen-presenting cells" or "APCs," as used herein, preferably refers to professional antigen-presenting cells (e.g., dendritic cells, macrophages, B cells, etc.) that are distinct from non-professional antigen-presenting cells (e.g., fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, vascular endothelial cells, etc.). In preferred embodiments, the APCs are professional antigen-binding cells, preferably endogenous professional APCs, of the subject in need of treatment. In certain embodiments, the endogenous professional APCs are not cells of the tumor or cancer being treated in the subject.

[0156] In either embodiment, the first antigen binding protein of the bispecific polypeptide specifically binds to an APC antigen selected from the group consisting of MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, CD206 (also known as the mannose receptor), 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; galectins are expressed by T helper cells and B cells; CD11c is expressed at high levels by DCs; CD19 is expressed on all professional 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. CD206 is primarily present on the surface of macrophages and immature dendritic cells. In any embodiment, the antigen specifically bound by the first binding protein is not a tumor-associated antigen.

[0157] In either embodiment, the second antigen-binding protein of the bispecific polypeptide specifically binds to an antigen on an immune effector cell expressing a CAR or similar receptor. For example, the second binding protein of a bispecific polypeptide for use in the methods of the invention can bind to an antigen on an immune cell, where the antigen is not part of the CAR but is present on the cell surface of the immune cell. Alternatively, the second antigen-binding protein can bind directly to a CAR expressed by an immune cell, for example, to the antigen-binding protein of the CAR or to an extracellular region of the CAR that is not involved in antigen binding, as described further below.

[0158] In certain embodiments, the CAR present on the immune cell may contain additional amino acids or molecules for binding by a second antigen-binding protein. As is known in the art, CAR constructs can be designed to contain a "tag," which is usually 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 engineered to express a CAR that contains a tag. In the context of such embodiments, the second antigen-binding protein of the bispecific polypeptide may bind to the tag or to a region of the CAR other than the tag.

[0159] Illustrative examples of tags that may be 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 proteins for binding to antibodies or other proteins, nucleoproteins (e.g., leucine zippers), or any other protein modification to the CAR). In either embodiment, the antigen or affinity tag on the CAR is a c-Myc-tag or a FLAG-tag.

[0160] In some embodiments, the immune cells are T cells or NK cells engineered to express a CAR that does not contain a tag. In some embodiments, the immune cells are T cells or NK cells engineered to express a CAR that does not contain a tag or any heterologous tumor-associated antigen or fragment of a tumor-associated antigen.

[0161] In certain embodiments, the immune cell is a T cell or NK cell engineered to express a CAR, and the bispecific polypeptide binds to the extracellular portion of the CAR. The extracellular domain of the CAR typically comprises an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen. 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 also typically 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, e.g., IgG1 or IgG4, or may be derived from an alternative cell surface protein, including, but not limited to, CD4, CD8, or CD28.

[0162] Thus, in certain embodiments, the second antigen-binding protein of a bispecific polypeptide for use in the methods of the invention may be for binding to the antigen-binding domain of a CAR. As such, the second antigen-binding protein of the bispecific polypeptide may be derived from an anti-idiotypic antibody or antigen-binding fragment thereof, where the anti-idiotypic antibody is an anti-idiotypic antibody of the antibody portion of the CAR.

[0163] Anti-idiotypic antibodies are known in the art, and one of skill in the art would be able to utilize the antigen-binding domains of these antibodies in designing bispecific polypeptides for use in the methods of the invention. Thus, in some embodiments, the second antigen-binding domain of a bispecific polypeptide for use in the methods of the invention comprises an antibody or antibody fragment derived from an anti-idiotypic antibody specific for anti-CD19, anti-Her2, anti-mesothelin, or other antibody, or an antigen-binding fragment of an anti-idiotypic antibody for binding to an antibody found in a CAR.

[0164] Some anti-idiotypic antibodies are known in the art. For example, International Patent Application Publication No. WO2014 / 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 is currently being used in several CAR constructs under development.

[0165] In certain embodiments, the second antigen-binding protein of a bispecific polypeptide for use in the methods of the invention comprises an antibody or antigen-binding protein 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 of the CDRs identical to mAb clone no. 136.20.1 (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). In some embodiments, the 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 mAb clone no. 136.20.1. In some embodiments, the second antigen binding protein 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 that binds to the same epitope as mAb clone no. 136.20.1.

[0166] Other examples of anti-idiotype antibodies include those commercially available from AbD Serotec®, the anti-CD22 antibody specific anti-idiotype antibody described in International Patent Publication No. WO 2013 / 188864, the anti-CEA antibody specific anti-idiotype antibody described in International Patent Publication No. WO 97 / 34636, the anti-GD2 antibody specific anti-idiotype antibody described in U.S. Patent No. 5,935,821, and the anti-NY-ESO-1 antibody specific anti-idiotype antibody described in Jakka et al. 2013, Anticancer Research 33(10): 4189-420. Custom anti-idiotype antibodies are also available from AbD Serotec®.

[0167] 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.

[0168] In further embodiments, the bispecific polypeptide comprises a second antigen-binding protein that binds to an extracellular region of the CAR that is not involved in antigen binding. For example, the second binding protein 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 to the spacer region between the variable heavy and variable light chains of the scFv portion of the CAR, or to any other region of the CAR.

[0169] In some embodiments, the hinge region may be an scFv-CD28 or scFv-CD8 junction and include a neo-epitope that can be targeted by a second binding protein. In some embodiments, the hinge region may include a mutated (Fe-binding null) IgG CH2 / 3 that can be targeted by a second binding protein. 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 protein.

[0170] An example of an anti-CAR antibody that binds to the hinge region of a CAR molecule is the 2D3 antibody described in International Patent Application Publication No. WO2014 / 190273, which binds to an IgG4 CH2-CH3 hinge region. In some embodiments, a multispecific antigen-binding construct comprises an antigen-binding polypeptide construct that binds to an IgG4 CH2-CH3 hinge region. In some embodiments, a multispecific antigen-binding construct comprises an antigen-binding polypeptide construct that binds to an IgG4 CH2-CH3 hinge region and has one or more of the CDRs identical to 2D3 described in WO2014 / 190273 (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. In some embodiments, the second binding protein of the bispecific polypeptide binds to the IgG4 CH2-CH3 hinge region and binds to the same epitope as 2D3 described in WO2014 / 190273.

[0171] In further embodiments, the second antigen-binding protein of the bispecific polypeptide binds to an antigen on an immune cell expressing a CAR, where the antigen is not the CAR or is not on the CAR. For example, the antigen can include naturally occurring proteins present on the surface of immune cells (e.g., CD3, CD4, CD8, CD25, CD127, CD196 (CCR6), CD27, CD28, CD45RA, CD45RO, CD62L, CD197, and HLA-DR). Alternatively, the antigen on the immune cell can 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).

[0172] The first and second antigen-binding proteins of the bispecific polypeptide for use in the methods of the present invention may be in the form of an antibody or antibody fragment. In one embodiment, the first antigen-binding protein comprises an scFv. In another embodiment, the second antigen-binding protein comprises an scFv. In certain embodiments, the first and second antigen-binding proteins are in different antibody or antibody fragment formats. Alternatively, the first and second antigen-binding proteins may be in the same antibody or antibody fragment format. In certain examples, the first antigen-binding protein (i.e., for binding to an antigen on an APC, preferably a professional APC) is in the form of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4) and the second antigen-binding protein is in the form of an antibody fragment (e.g., scFv). In a further example, the second antigen binding protein (i.e., for binding to an antigen on an immune cell, preferably a CAR T cell) is in the form of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4) and the first antigen binding protein (for binding to an antigen on an APC) is in the form of an antibody fragment (e.g., an scFv).

[0173] In a preferred embodiment, the bispecific polypeptide for use according to the present invention is in the form of a fusion protein. The fusion protein preferably comprises a first antigen-binding protein joined, either directly or via a linker, to a second antigen-binding protein. The linker may be any linker known to those skilled in the art for use in the context of fusion proteins.

[0174] A linker can function, for example, to link two proteins of an antigen-binding polypeptide construct (e.g., the VH and VL of an scFv or diabody), or to link two antigen-binding polypeptide constructs together (e.g., two or more Fabs or sdAbs), or to link an antigen-binding polypeptide construct to a scaffold. In some embodiments, a bispecific polypeptide can include multiple linkers (i.e., two or more), for example, one or more scFvs linked to a scaffold can include 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 the skilled artisan based on the intended use of the linker (see, e.g., Miller & Kontermann, "Bispecific Antibodies" in Handbook of Therapeutic Antibodies, Wiley-VCR Verlag GmbH & Co. 2014).

[0175] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art and contain glycine and serine units in various combinations. Examples include, but are not limited to, (GS), (GSGGS), (GGGS), and (GGGGS), where n is an integer of at least 1 and is typically between 1 and about 10, e.g., between 1 and about 8, between 1 and about 6, or between 1 and about 5.

[0176] Other useful linkers include sequences derived from immunoglobulin hinge sequences. The linker may contain all or part of a hinge sequence derived from any one of the four IgG classes, and may optionally contain additional sequences. For example, the linker may contain a portion of an immunoglobulin hinge sequence and a glycine-serine sequence. A non-limiting example is a linker containing approximately the first 15 residues of an IgG1 hinge, followed by a GlySer linker sequence about 10 amino acids in length, such as those described above.

[0177] The length of the linker varies depending on the application. A suitable linker length can be readily selected by one skilled in the art. For example, when the linker connects the VH and VL domains of an scFv, the linker is typically between about 5 and about 20 amino acids in length, e.g., between about 10 and about 20 amino acids in length, or between about 15 and about 20 amino acids in length. When the linker is intended to connect the VH and VL domains of a diabody, the linker must be short enough to prevent association of these two domains within the same chain. For example, the linker can be between about 2 and about 12 amino acids in length, e.g., between about 3 and about 10 amino acids in length, or about 5 amino acids in length.

[0178] In some embodiments, when the linker is intended to connect two Fab fragments, the linker can be selected to maintain the relative spatial conformation of the paratopes of the F(ab') fragments and to be capable of forming a covalent bond equivalent to the disulfide bond in the core hinge of IgG. In this context, suitable linkers include those derived from IgG hinge regions, such as 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).

[0179] The linker may comprise a sequence of amino acid residues connecting the first and second antigen-binding proteins. Alternatively, the first and second binding proteins may be linked via chemical conjugation (e.g., to form a bis-aryl conjugate between the proteins). Examples of suitable methods for chemical conjugation of binding proteins are known in the art. Such methods include the use of succinimidyl compound modification of primary amines present on lysine residues, such as those used in TriLink Technologies bioconjugation reagents. In one embodiment, when the first and / or second binding proteins are scFvs, the bispecific polypeptide comprises a linker sequence between the VH and VL proteins of the scFv. Suitable linker sequences are known to those of skill in the art, and illustrative examples include relatively flexible, hydrophilic amino acid residues.

[0180] It will be within the skill of the art to determine the appropriate structure of a bispecific polypeptide for use in accordance with the present invention.

[0181] In either embodiment, the first and / or second binding domain may be in the form of an antibody or an antigen-binding fragment thereof. The antigen-binding protein may be an antibody, for example, a monoclonal antibody. The antigen-binding protein may be in the form of a recombinant antibody or a modified antibody (e.g., a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a primatized antibody, a deimmunized antibody, a synhumanized antibody, a half antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody). The antibody may further comprise chemical modifications, for example, conjugation to an active substance or radiolabel or an agent to improve solubility, or other modifications described herein. As used herein, an antigen-binding protein may be a variable domain.

[0182] The first and second antigen-binding proteins of the bispecific polypeptide are preferably in the form of an antibody or antigen-binding fragment thereof. In either embodiment, the first and / or second antigen-binding protein is in the form of an antibody, for example, immunoglobulin G (IgG1, IgG2, IgG3, or IgG4). Optionally, the first antigen-binding protein is in the form of an antibody, for example, immunoglobulin G (IgG1, IgG2, IgG3, or IgG4), and the second antigen-binding protein is in the form of an antibody fragment (e.g., scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2), and the second antigen-binding protein is linked to the first antigen-binding protein via the C- or N-terminus or via an internal region of the first antigen-binding protein, preferably via the C-terminus of the heavy chain of the first antigen-binding protein. Optionally, the second antigen binding protein is in the form of an antibody, for example immunoglobulin G (IgG1, IgG2, IgG3 or IgG4), and the first antigen binding protein is in the form of an antibody fragment (e.g. scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2), and the first antigen binding protein is linked to the second antigen binding protein via the C- or N-terminus or via an internal region of the second antigen binding protein, preferably the second antigen binding protein is linked to the second antigen binding protein via the C-terminus of the heavy chain of the second antigen binding protein.

[0183] In any embodiment, a bispecific polypeptide for use according to the present invention may contain additional amino acids or molecules for purification or identification. For example, the polypeptide may contain an epitope or affinity tag. Illustrative 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.

[0184] In either embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD40. In either embodiment, the bispecific polypeptide comprises a second antigen binding protein for binding to a FLAG tag.

[0185] In either embodiment, the bispecific polypeptide comprises a first antigen binding protein and a second antigen binding protein, wherein the first antigen binding protein specifically binds to CD40 and the second antigen binding protein specifically binds to a FLAG tag.

[0186] Preferably, the first antigen binding protein binds to the outer A module of the membrane proximal domain (D1a) of CD40, and optionally, said binding does not prevent, reduce or inhibit CD40-CD40L binding.

[0187] In one embodiment, the first antigen binding protein is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, an antigen-binding domain comprising where: FR1, FR2, FR3, and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a, and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; The sequences of any framework regions and / or complementarity determining regions are as described herein, preferably as described in Table 1 in the context of a CD40 binding protein.

[0188] In either embodiment, CDR1, CDR2, and CDR3 refer to the complementarity determining regions from the variable heavy chain (VH) of the antibody, and CDR1a, CDR2a, and CDR3a are the complementarity determining regions of the variable light chain (VL) of the antibody, or where CDR1, CDR2, and CDR3 are the complementarity determining regions from the VL, CDR1a, CDR2a, and CDR3a are the complementarity determining regions from the VH. In such instances, the CDRs may sometimes be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0189] Reference herein to a protein or antibody that "binds" to CD40 provides literal support for the protein or antibody "binds specifically to" or "specifically binds to" CD40.

[0190] In either embodiment, the first antigen binding protein competitively inhibits the binding to CD40 of an antibody or antigen-binding fragment thereof comprising a VH comprising the sequence set forth in SEQ ID NO:1 and a VL comprising the sequence set forth in SEQ ID NO:2.

[0191] In either embodiment, the first antigen binding protein comprises antigen binding domains CDRH1, CDRH2 and / or CDRH3 having variable heavy domains as defined in SEQ ID NO:1.

[0192] In either embodiment, the first antigen binding protein comprises antigen binding domains CDRL1, CDRL2 and / or CDRL3 having a light chain variable domain as defined in SEQ ID NO:2.

[0193] In either embodiment, the first antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain defined in SEQ ID NO:1 and a variable light chain defined in SEQ ID NO:2.

[0194] In any embodiment, the first antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, wherein the sequences of any of the framework regions and / or complementarity determining regions are as described herein, and preferably as described in Table 1 below.

[0195] As defined herein, a linker can be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0196] In any embodiment, the first antigen binding protein is (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 4, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 5; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 6, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 8; or (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO:5; and (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 6; a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 8; Includes. In any embodiment, the first antigen binding protein is (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 9, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 10, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 11; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 13, and a DR3 comprising or consisting of the sequence set forth in SEQ ID NO: 8; or (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:9, or at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the sequence set forth in SEQ ID NO:10. , or a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 11; (iv) a CDR1 comprising or consisting of a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 12; a CDR2 comprising, or consisting of, a sequence that is at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:8; a CDR3 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:8; Includes.

[0197] In either embodiment, FR1, FR2, FR3, and FR4 may refer to framework regions from the variable heavy chain (VH) of an antibody, and FR1a, FR2a, FR3a, and FR4a may refer to framework regions from the variable light chain (VL) of an antibody, or if FR1, FR2, FR3, and FR4 are framework regions from a VL, then FR1a, FR2a, FR3a, and FR4a are framework regions from a VH. In such examples, the FRs may sometimes be referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3, and FR L4.

[0198] In one embodiment, the second antigen binding protein comprises an antigen binding domain having FR H1, FR H2, FR H3 and / or FR H4 of the antigen binding domain with a variable heavy chain as defined in SEQ ID NO: 1 (according to Kabat or IMGT numbering).

[0199] In either embodiment, the second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 of the antigen binding domain with a variable light chain as defined in SEQ ID NO:2 (according to Kabat or IMGT numbering).

[0200] In either embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable heavy chain defined in SEQ ID NO:1 (according to Kabat or IMGT numbering), and FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable light chain defined in SEQ ID NO:2 (according to Kabat or IMGT numbering).

[0201] In some embodiments, the first antigen binding protein comprises a heavy chain variable domain comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 1, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto. Optionally, the heavy chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence as set forth in SEQ ID NO: 1, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD40.

[0202] In some embodiments, the first antigen binding protein comprises a light chain variable domain comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO:2, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto. Optionally, the light chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence as set forth in SEQ ID NO: 2, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD40.

[0203] In any embodiment, the first antigen binding protein comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 1, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto. and a light chain variable domain comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO:2, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Optionally, the heavy and / or light chain variable domain of the first antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19 or no more than 20 amino acid residue substitutions compared to the amino acid sequence as set forth in SEQ ID NO: 1 or 2, respectively, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to CD40.

[0204] In either embodiment, the first antigen binding protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 1 and 2 (in N to C-terminal or C to N-terminal order).

[0205] In any embodiment, the first antigen binding protein may have, from N to C terminus, a VH then a VL, or a VL then a VH, or any of CDR1, 2 and 3 defined herein as VH then any of CDR1, 2 and 3 defined herein as VL, or any of CDR1, 2 and 3 defined herein as VL then any of CDR1, 2 and 3 defined herein as VH.

[0206] In any embodiment, the first antigen binding protein may be in the form: (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) a dimeric scFv (di-scFv), or (iv) one of (ii) or (iii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0207] Furthermore, as described herein, the first antigen binding protein may be in the form: (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, (vii) other forms of bispecific or multispecific antibodies; (viii) one of (i) to (vii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0208] In any embodiment, the first antigen-binding protein may be in the form of an immunoglobulin G molecule (IgG). Optionally, according to any embodiment of the first aspect of the invention, the first antigen-binding protein may be in the form of an IgG or heterodimeric Fab-Fc, and the second antigen-binding protein may be in the form of an antigen-binding fragment of IgG, for example an scFv.

[0209] In any embodiment, the second antigen binding protein is for binding to a FLAG tag, and the second antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a an antigen-binding domain comprising where: FR1, FR2, FR3, and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a, and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; The sequences of any framework regions and / or complementarity determining regions are as described herein, and preferably as set out in Table 1 below.

[0210] In either embodiment, CDR1, CDR2, and CDR3 refer to the complementarity determining regions from the heavy variable chain (VH) of the antibody, and CDR1a, CDR2a, and CDR3a are the complementarity determining regions from the variable light chain (VL) of the antibody, or if CDR1, CDR2, and CDR3 are the complementarity determining regions from the VL, then CDR1a, CDR2a, and CDR3a are the complementarity determining regions from the VH. In such instances, the CDRs may sometimes be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0211] In either embodiment, the second antigen binding protein is capable of specifically binding to a FLAG tag or a variant thereof (such as defined in SEQ ID NO: 28 or 30, or as otherwise defined herein). The antigen binding protein is preferably capable of specifically binding to a protein domain comprising multiple FLAG tag sequences (e.g., 2xFLAG, 3xFLAG, etc.).

[0212] In either embodiment, the second antigen binding protein is for specifically binding to a peptide tag containing, or consisting of, the sequence DYK, preferably the sequence DYKD (SEQ ID NO: 28). In addition to these sequences, other amino acids may be present in the FLAG tag bound by the second antigen binding protein; preferably, the additional amino acids in the FLAG tag are hydrophilic amino acids, such as R (Arg), D (Asp), E (Glu) and K (Lys), and / or amino acids with aromatic side chains, such as Y (Tyr), F (Phe), H (His) and W (Trp).

[0213] In a preferred embodiment, the second antigen-binding protein is capable of specifically binding to a FLAG-tag containing, comprising, or consisting of the sequence GDYKDDDDKG (SEQ ID NO:29), DYKDDDDK (SEQ ID NO:30), MDYKDDDDK (SEQ ID NO:31), DFKDDDK (SEQ ID NO:32), DYKAFDNL (SEQ ID NO:33), DYKDHDG (SEQ ID NO:34), MDFKDDDDK (SEQ ID NO:35), MDYKAFDNL (SEQ ID NO:36), DYKDHDI (SEQ ID NO:37), DYKDH (SEQ ID NO:38), DYKDD (SEQ ID NO:39), DYKDHD (SEQ ID NO:40) and / or DYKDDD (SEQ ID NO:41). The most preferred sequence is DYKDDDDK (SEQ ID NO:30).

[0214] As used herein, the term FLAG-tag also encompasses modified FLAG tags, for example those derived from the above-mentioned FLAG tags, in particular tags having the sequence DYKDDDDK, by amino acid insertion, deletion or substitution.

[0215] Reference herein to a protein or antibody that "binds" a FLAG tag provides literal support for the protein or antibody that "binds specifically to" or "specifically binds to" the FLAG tag.

[0216] In either embodiment, the FLAG tag is present at the N-terminus, C-terminus or within the protein to which the bispecific polypeptide of the invention is capable of binding, binds or specifically binds.

[0217] In either embodiment, the second antigen binding protein competitively inhibits binding to a FLAG tag of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 15 and a VL comprising the sequence set forth in SEQ ID NO: 16.

[0218] In either embodiment, the second antigen binding protein comprises an antigen binding domain CDRH1, CDRH2 and / or CDRH3 having a variable heavy chain as defined in SEQ ID NO:15.

[0219] In either embodiment, the second antigen binding protein comprises antigen binding domains CDRL1, CDRL2 and / or CDRL3 having a light chain variable domain as defined in SEQ ID NO:16.

[0220] In embodiments of any of the second aspect, the second antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain defined in SEQ ID NO: 15 and a variable light chain defined in SEQ ID NO: 16.

[0221] In any embodiment, the second antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, wherein the sequences of any of the framework regions and / or complementarity determining regions are as described herein, and preferably as described in Table 1.

[0222] As defined herein, a linker can be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0223] In any embodiment, the second antigen-binding domain comprises: (a) a CDR1 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17; a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 19; and (b) a CDR1 comprising, or consisting of, an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 20; a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 22; or (c) a complementarity determining region CDR1 comprising or consisting of an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 23, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VH comprising a CDR2 comprising, or consisting of, an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO:25; and (d) a CDR1 comprising or consisting of an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 26; a CDR2 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 22; The antigen-binding domain comprises:

[0224] In any embodiment, the second antigen binding protein is (a) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 19; and (b) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22; or (c) a VH comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; and (d) a VL comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22 The antigen-binding domain comprises:

[0225] In either embodiment, FR1, FR2, FR3, and FR4 may refer to framework regions from the variable heavy chain (VH) of an antibody, and FR1a, FR2a, FR3a, and FR4a may refer to framework regions from the variable light chain (VL) of an antibody, or FR1, FR2, FR3, and FR4 are framework regions from the VL and FR1a, FR2a, FR3a, and FR4a are framework regions from the VH. In such examples, the FRs are sometimes referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3, and FR L4.

[0226] In either embodiment, the second antigen binding protein comprises an antigen binding domain having a FR H1, FR H2, FR H3 and / or FR H4 derived from human germline, wherein the human germline is IGHV1-46 * 01 or IGHV7-4-1 * The number is 02.

[0227] In either embodiment, the second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 derived from human germline, wherein the human germline is selected from the group consisting of IGKV2-30, ... * 01 or IGKV4-1 * The number is 01.

[0228] In either embodiment, the second antigen binding protein is IGHV1-46 *01 or IGHV7-4-1 * FR H1, FR H2, FR H3 and / or FR H4 derived from a human germline, and IGKV2-30 * 01 or IGKV4-1 * 01.

[0229] In one embodiment, the second antigen binding protein comprises an antigen binding domain having FR H1, FR H2, FR H3 and / or FR H4 of the antigen binding domain having a variable heavy chain as defined in SEQ ID NO: 15 (according to Kabat or IMGT numbering).

[0230] In either embodiment, the second antigen binding protein comprises an antigen binding domain having FR L1, FR L2, FR L3 and / or FR L4 of the antigen binding domain with a variable light chain as defined in SEQ ID NO: 16 (according to Kabat or IMGT numbering).

[0231] In either embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable heavy chain defined in SEQ ID NO: 15 (according to Kabat or IMGT numbering) and FR1, FR2, FR3 and / or FR4 of an antigen binding domain having a variable light chain defined in SEQ ID NO: 16 (according to Kabat or IMGT numbering).

[0232] In some embodiments, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto. Optionally, the heavy chain variable domain of the second antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 15, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to a FLAG tag.

[0233] In some embodiments, the second antigen binding protein comprises an antigen binding domain comprising a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto. Optionally, the light chain variable domain of the second antigen binding protein comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequence set forth in SEQ ID NO: 16, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to a FLAG tag.

[0234] In some embodiments, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15 and a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Optionally, the heavy and light chain variable domains of the second antigen binding protein comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, or no more than 20 amino acid residue substitutions compared to the amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively, optionally none of the amino acid substitutions are in the CDRs, and / or the antigen binding protein retains the ability to bind to a FLAG tag.

[0235] As described herein, the second antigen binding protein may be in the form of: (i) single domain antibodies (sdAbs), (ii) single chain Fv fragment (scFv), (iii) dimeric scFv (di-scFv), (iv) one of (ii) or (iii) linked to a constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; (v) one of (i) to (iv) linked to a protein that binds to an immune effector cell; (vi) one of (i) to (iv) linked to a modified immune cell receptor, e.g., a modified T cell receptor.

[0236] Furthermore, as described herein, the second antigen binding protein may be in the form: (i) diabodies, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, (vii) other forms of bispecific or multispecific antibodies; (viii) one of (i) to (vii) linked to the constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0237] In a preferred embodiment, the second antigen-binding protein is in the form of an scFv and the first antigen-binding protein is in the form of an immunoglobulin G (IgG) antibody.

[0238] In any embodiment of any aspect of the invention, the bispecific polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 42 and 43.

[0239] As used herein, the complementarity determining region sequences (CDRs) of any antigen binding protein referred to herein are defined according to the IMGT, Chothia or Kabat numbering systems or any other CDR numbering system.

[0240] Nucleic acids and vectors In another aspect disclosed herein, there are provided nucleic acids encoding the bispecific polypeptides for use in the methods described herein. In yet another aspect, there are provided cells comprising the vectors for use in the methods described herein. For example, it will be appreciated that in certain embodiments, the bispecific polypeptides are not administered directly to a subject in need of treatment, but instead may be expressed in vivo in the subject from a nucleic acid sequence or construct provided to the subject.

[0241] The terms "polynucleotide," "polynucleotide sequence," "nucleotide sequence," "nucleic acid," or "nucleic acid sequence" are used interchangeably herein to refer to mRNA, RNA, cRNA, cDNA, or DNA. The term refers to a polymeric form of nucleotides, usually at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes single- and double-stranded forms of RNA and DNA.

[0242] In another aspect, a vector is provided comprising a nucleic acid described herein operably linked to a regulatory sequence.

[0243] The term "regulatory element" or "regulatory sequence" refers to a nucleic acid sequence (e.g., DNA) necessary for the expression of an operably linked coding sequence in a particular cell. Regulatory sequences that are suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailing sequences that control mRNA stability, and targeting sequences that target the product encoded by the transcribed polynucleotide to an intracellular compartment within the cell or to the extracellular environment.

[0244] Typically, regulatory sequences include, but are not limited to, 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, translation initiation sites and / or nucleotide sequences encoding leader sequences, stop codons, translation stop sites, and 3' non-translated regions. Constitutive or inducible promoters known in the art are contemplated. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter.

[0245] The contemplated promoter sequences may be native to mammalian cells or may be derived from alternative sources, with the region being functional in the selected organism. The choice of promoter will depend on the intended host cell. For example, promoters that may be used for expression in mammalian cells include, among others, the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, the □-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 LTR promoter, the mouse mammary tumor virus 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 well described in the art and are readily available.

[0246] Enhancer elements can also 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 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 in Boshart et al. (1985, Cell, 41:521).

[0247] The vector construct may also contain 3' non-translated sequences. The term "3' non-translated sequence" refers to a portion of a gene, including a DNA segment containing a polyadenylation signal and any other regulatory signals capable of achieving mRNA processing or gene expression. A 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 their homology to the canonical form 5'AATAAA-3', although variations are not uncommon. The 3' non-translated regulatory DNA sequence preferably contains 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 achieving mRNA processing or gene expression.

[0248] The terms "operably connected" or "operably linked," as used herein, refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence "operably linked" to a coding sequence refers to the positioning and / or orientation of the regulatory sequence relative to the coding sequence that permits expression of the coding sequence under conditions compatible with the regulatory sequence.

[0249] 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. The types of vectors and techniques for inserting nucleic acid constructs for use in accordance with the methods of the present invention are known in the art, and illustrative examples include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0250] Methods for producing bispecific polypeptides Bispecific polypeptides for use in the methods described herein can be produced using any number of expression systems that would be known to one of skill in the art, illustrative examples of which include 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 one of skill in the art, illustrative examples of which include CHO or 293 expression systems. These expression systems are widely available from proprietary suppliers. In one embodiment, the bispecific polypeptides are produced using a mammalian expression system.

[0251] Chemically conjugated bispecific polypeptides can be formed following succinimidyl compound modification of primary amines present on lysine residues as described elsewhere herein.

[0252] It would be within the skill of the art to utilize commercially available antibodies or antigen-binding domains thereof to generate bispecific polypeptides for use in the methods described herein.

[0253] Furthermore, it is within the skill of the art to recreate an antibody or antigen-binding protein thereof based on published sequence information for an antibody or antigen-binding protein with the desired specificity, and thereby include such an antibody or antigen-binding protein thereof in a bispecific polypeptide for use in accordance with the methods of the invention. In particular, it will be understood that the methods of the invention encompass the production of any bispecific polypeptide designed to have the specific binding affinity of the first and second antigen-binding proteins described herein (i.e., a first binding protein for binding to an antigen on an antigen-presenting cell, preferably a professional antigen-presenting cell, e.g., a dendritic cell, and a second binding protein for binding to an antigen on an immune cell expressing a CAR, including when the antigen is an antigen on a CAR). It will be understood by those skilled in the art that any combination of antigen-binding proteins with the desired binding specificities can be utilized to obtain the bispecific polypeptide of the invention.

[0254] Furthermore, it is within the skill set of a person skilled in the art to evaluate the binding capacity of any bispecific polypeptide prepared for use according to prior art methods. For example, methods for evaluating protein binding are known in the art, as described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing an antigen-binding protein and contacting it with a labeled antigen. After washing to remove non-specifically bound proteins, the amount of label, and thus the amount of bound antigen, is detected. Of course, the antigen-binding protein can also be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or in addition, surface plasmon resonance assays can be used.

[0255] Optionally, the dissociation constant (Kd), association constant (Ka), and / or affinity constant (KD) of the immobilized antigen-binding protein is determined. The "Kd" or "Ka" or "KD" of a bispecific polypeptide for use according to the present invention is measured, in one example, by a radiolabeled or fluorescently labeled ligand binding assay. In the case of "Kd," the assay equilibrates the antigen-binding protein with a minimum concentration of labeled antigen in the presence of a titration series of unlabeled antigen. After washing to remove unbound antigen, the amount of label is determined, which indicates the Kd of the protein.

[0256] K d According to another example from K. a or K D is measured using a surface plasmon resonance assay, for example, using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized antigen or immobilized antigen-binding protein.

[0257] Pharmaceutical Composition In another aspect, there is provided a pharmaceutical composition comprising a bispecific polypeptide as described herein and a pharmaceutically acceptable carrier for use according to any of the methods of the invention.

[0258] The compositions described for use herein may be prepared by methods known in the art and are suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition together with one or more pharmaceutically acceptable carriers or diluents.

[0259] The term "pharmaceutically acceptable carrier," as used herein, refers to any suitable carrier, diluent, or excipient. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, and solutes which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents, etc. It will be understood that the compositions for use in the methods of the present invention may also contain other supplementary physiologically active substances.

[0260] Carriers are generally pharmaceutically "acceptable" in the sense that they are compatible with other ingredients in the composition and are not harmful to the subject. Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous, and intradermal administration. The compositions may conveniently be presented in unit dosage form and can be prepared by any method known in the art of pharmacy. Such methods include preparing a carrier for association with isolated T cells. Generally, the compositions are prepared by uniformly and intimately associating any active ingredient with a liquid carrier.

[0261] In one embodiment, the composition is suitable for parenteral administration, hi another embodiment, the composition is suitable for intravenous administration.

[0262] 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.

[0263] The present disclosure also contemplates the compositions described herein in combination 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 known active agents, the combination can be administered either sequentially (either continuously or separated by periods of no treatment) or simultaneously or as a mixture. Suitable anti-cancer agents will be known to those skilled in the art. Combined treatments are also contemplated to include a composition for use in the methods of the present invention followed by a known treatment, or treatment with a known agent followed by treatment with either a composition for use in the methods of the present invention, for example, as maintenance therapy.For example, in the treatment of cancer, compositions for use in the methods of the invention may comprise alkylating agents (e.g., mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin or platinum-containing alkylating agents, e.g., cisplatin, carboplatin, and oxaliplatin) and antimetabolites (e.g., purine or pyrimidine analogs or antifolates, e.g., azathioprine and mercaptopurine), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin idarubicin, valrubicin, mitoxantrone or anthracycline analogues), plant alkaloids (e.g., vinca alkaloids or taxanes, e.g., vincristine, vinblastine, vinorelbine, vindesine, paclitaxel or doestaxel), topoisomerase inhibitors (e.g., type I or type II topoisomerase inhibitors), podophyllotoxins (e.g., etoposide or teniposide), Tyrosine kinase inhibitors (e.g., imatinib mesylate, nilotinib, or dasatinib), adenosine receptor inhibitors (e.g., A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP, or A2BR inhibitors, e.g., PSB-1115), adenosine receptor agonists (e.g., CCPA, IB-MECA, and CI-IB-MECA), those of the PDL-1:PD-1 axis, nivolumab, pembrolizumab, atezolizumab , BMS-936559, MEDI4736, MPDL33280A or MSB0010718C), inhibitors of the CTLA-4 pathway (e.g., ipilimumab and tremelimumab), inhibitors of the TIM-3 pathway, or agonist monoclonal antibodies known to promote T cell function (including anti-OX40, e.g., MEDI6469, and anti-41BB, e.g., PF-05082566).

[0264] In a further aspect, there is provided a kit or article of manufacture for use in the methods of the invention, the kit or article of manufacture of the invention comprising one or more bispecific polypeptides of the invention, a nucleic acid encoding said bispecific polypeptides and / or a pharmaceutical composition as described above.

[0265] In a further aspect, there is provided a kit for use in the methods of the invention, the kit comprising: (a) a container holding a bispecific polypeptide, nucleic acid, vector or pharmaceutical composition of the invention; and (b) the label or package insert containing instructions for use; Includes:

[0266] The kit may also include one or more active ingredients or components for the treatment of cancer. For example, the kit may also include immune cells expressing a CAR.

[0267] A "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 a 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 to treat 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.

[0268] The kit may include (a) a therapeutic or prophylactic composition and (b) a second container containing a second active ingredient or components. A kit according to this embodiment for use in the methods of the invention may further include a package insert indicating that the composition and other active ingredients can be used to treat cancer or a condition described herein. Alternatively, or in addition, the kit may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. Other materials desirable from a commercial and user standpoint, as would be known to those skilled in the art, may further be included; suitable examples of which include other buffers, diluents, filters, needles, and syringes. [Example]

[0269] Example 1 Bispecific BEAT and multiple stimulation of CAR T cells with either MoDC or tumor A bispecific polypeptide (i.e., anti-CD40 / anti-FLAG BEAT) comprising a first antigen-binding protein for binding to CD40 and a second antigen-binding protein for binding to a FLAG tag was obtained according to the methods and disclosures herein. The amino acid sequence of the BEAT is provided in SEQ ID NOs: 42 and 43.

[0270] CAR T cells were obtained by transducing CD3+ T cells with a nucleic acid sequence encoding a CAR containing a FLAG-tag.

[0271] CAR T cells were stimulated with either high-expressing mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs) (Figure 1). Additional stimulators (tumor or MoDCs) were added on days 2 and 4 for each condition. Activation and exhaustion markers were measured on day 6 (Figure 2). Phenotypes of all conditions were also assessed (Figure 3).

[0272] The CAR T cell + BEAT and MoDC groups showed reduced CAR T cell exhaustion markers LAG-3, PD-1, and Tim-3 compared with tumor-primed CAR T cells and even after multiple stimulations with MoDCs. In comparison, the CAR T cell + tumor group treated with multiple tumor stimulations on days 2 and 4 had increased activated (CD25, CD69) and exhausted (LAG-3, PD-1, and Tim-3) phenotypes. Activation and exhaustion were not prevalent in a 6-day proliferation assay using the CAR + tumor group that did not receive multiple tumor stimulations (Figure 2).

[0273] Furthermore, restimulation of CAR T cells with BEAT and MoDC did not alter the CAR T cell memory phenotype (TSCM and TCM) compared with CAR T cells without BEAT and MoDC boost. However, the CAR T cell phenotype after tumor restimulation showed an increased effector memory phenotype (TEM) and decreased TSCM compared with CAR T cells without additional tumor stimulation. The TCM and TSCM CAR T cell phenotypes were also similar for the CD4+ and CD8+ subsets between CAR T cells with tumor stimulation (without restimulation) and CAR T cells with BEAT and MoDC, both with and without restimulation, whereas the TSCM phenotype was increased in the CD8+ subset after stimulation (and restimulation) with BEAT and MoDC (Figure 3).

[0274] Example 2 Bispecific BEAT rescues exhausted tumor-pretreated CAR T cells In this experiment, CAR T cells were selected from each stimulation condition described in the examples above and restimulated with BEAT and MoDCs, either mesothelin-positive or mesothelin-negative tumors (Figure 4).

[0275] The phenotypes of all conditions were evaluated after 5 days of culture (day 11) (Figure 5). When CAR T cells were treated with mesothelin-positive tumors, BEAT- and MoDC-pretreated CAR T cells exhibited higher central memory (TCM) and stem cell memory (TSCM) phenotypes than tumor-pretreated CAR T cells, which had a higher effector memory phenotype (TEM). Surprisingly, restimulation of tumor-pretreated CAR T cells with BEAT and MoDC rescued the TCM phenotype (Figure 5B). In contrast, mesothelin-positive tumor restimulation of BEAT- and MoDC-pretreated CAR T cells enhanced the TCM and TSCM phenotypes (Figure 5A) compared with CAR T cells pretreated with and restimulated with mesothelin-positive tumors (Figure 5B).

[0276] Furthermore, BEAT- and MoDC-pretreated CAR T cells (Figure 6B) showed increased CAR T cell proliferation and signs of exhaustion compared with tumor-pretreated CAR T cells (Figure 6A). Restimulation with mesothelin-positive tumor or MoDC alone did not affect proliferation of tumor-pretreated CAR T cells. Proliferation was observed in BEAT- and MoDC-pretreated CAR T cells after restimulation with BEAT and MoDC or mesothelin-positive tumor antigen, but not in the presence of mesothelin-negative tumor (Figure 6).

Claims

1. 1. A method for rescuing immune effector cell activity or reversing immune effector cell exhaustion, comprising exposing immune effector cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby rescuing immune effector cell activity or reversing immune effector cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second antigen-binding protein that specifically binds to an antigen on an immune effector cell; - Optionally, the immune effector cells express a heterologous receptor comprising an antigen-binding protein and a signaling protein.

2. 1. A method for increasing immune effector cell activity or cytotoxicity of exhausted immune effector cells, comprising exposing a plurality of immune effector cells that exhibit one or more symptoms of an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby increasing immune cell effector activity or cytotoxicity; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell; - Optionally, the immune effector cells express a heterologous receptor comprising an antigen-binding protein and a signaling protein.

3. 1. A method for preventing, inhibiting, reducing or delaying the onset of immune effector cell exhaustion, comprising exposing a plurality of immune effector cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide, thereby preventing, inhibiting, reducing or delaying the onset of immune effector cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell; - Optionally, the immune effector cells express a heterologous receptor comprising an antigen-binding protein and a signaling protein.

4. 1. A method for treating a disease or condition characterized by immune effector cell exhaustion, comprising administering to a subject in need thereof an effective amount of a bispecific polypeptide, thereby treating the disease or condition characterized by immune effector cell exhaustion; - the bispecific polypeptide comprises a first antigen-binding protein that specifically binds to an antigen expressed on an antigen-presenting cell (APC), preferably a professional APC, and a second binding antigen-binding protein that specifically binds to an antigen on an immune effector cell of the subject.

5. The method of any one of claims 1 to 4, wherein the immune effector cell is a T cell, an NK cell, an NKT cell, or a γδT cell.

6. 6. The method of any one of claims 1 to 5, wherein the effector immune cells are T cells.

7. 7. The method of any one of claims 1 to 6, wherein the immune effector cells express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) that binds to a target antigen (e.g., a tumor-associated antigen).

8. 8. The method of any one of claims 1 to 7, wherein the immune effector cell is a CAR T cell and the bispecific polypeptide is for binding to an antigen on the CAR T cell.

9. 2. The method of claim 1, wherein the immune effector cells are CAR T cells and the method is for rescuing CAR T cell activity or reversing CAR T cell exhaustion.

10. 3. The method of claim 2, wherein the immune effector cells are CAR T cells and the method is for increasing CAR T cell activity or cytotoxicity of exhausted CAR T cells, preferably wherein the cells have or are suspected of having an exhausted phenotype.

11. 4. The method of claim 3, wherein the immune effector cells are CAR T cells and the method is for preventing, inhibiting, reducing or delaying the onset of CAR T cell exhaustion, preferably the cells having or suspected of having an exhausted phenotype.

12. 12. The method of any one of claims 1 to 11, comprising reversing at least one marker or sign of an exhaustion phenotype in immune effector cells.

13. 13. The method of any one of claims 1 to 12, wherein the step of exposing the immune effector cells to the bispecific polypeptide comprises contacting a population of immune effector cells obtained from the subject ex vivo with an effective amount of the bispecific polypeptide.

14. 14. The method of claim 13, further comprising administering to the subject the immune effector cells contacted with the bispecific polypeptide.

15. 13. The method of any one of claims 1 to 12, wherein the step of exposing immune cells to an effective amount of the bispecific polypeptide comprises administering the bispecific polypeptide to a subject in need of rescue or reversal of immune effector cell exhaustion, or increasing the activity of exhausted immune effector cells, or preventing, inhibiting, reducing or delaying the onset of immune effector cell exhaustion.

16. In the subject, - rescue immune effector cell (e.g., CAR T cell) activity; - Reverse immune effector cell (e.g., CAR T cell) exhaustion, - Increase the activity or cytotoxicity of exhausted immune effector cells (e.g., CAR T cells), or - a method of preventing, inhibiting, reducing, or delaying the development of immune effector cell (e.g., CAR T cell) exhaustion, comprising: administering to a subject a bispecific polypeptide; A method wherein the bispecific polypeptide comprises a first antigen binding protein that specifically binds to an antigen expressed on an antigen presenting cell (APC), preferably a professional APC, and a second binding antigen binding protein that specifically binds to an antigen on an immune (e.g., CAR T) cell.

17. 17. The method of any one of claims 1 to 16, wherein the bispecific polypeptide is administered to the subject after the subject has received treatment with CAR T cells or other immune effector cell treatment.

18. 18. The method of claim 17, wherein the bispecific polypeptide is administered to the subject at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or at least 6 weeks or longer after administration of the CAR T cells (or other immune effector cell treatment) to the subject.

19. 19. The method of any one of claims 1 to 18, wherein, at the time of administration of the bispecific polypeptide to the subject, one or more exhausted phenotypes of immune effector cells or markers or parameters indicative thereof are detectable or measurable in the subject or in a biological sample obtained from the subject.

20. 20. The method of any one of claims 1 to 19, wherein at the time of administration of the bispecific polypeptide to the subject, one or more exhausted phenotypes of immune effector cells or markers or parameters indicative thereof have been detected or measured in the subject or in a biological sample obtained from the subject.

21. 20. The method of any one of claims 1 to 19, comprising determining the risk or likelihood that a subject has immune effector cells (e.g., CAR T cells) with an exhausted phenotype.

22. 21. The method of claim 20, wherein determining whether the subject has or is at risk of having immune effector cells (e.g., CAR T cells) with an exhausted phenotype comprises determining the expression of one or more genes associated with immune cell exhaustion, and / or determining an increased level or degree of surface expression of markers on immune cells, and / or determining the percentage of immune cells that exhibit surface expression of one or more exhaustion markers in a sample of immune effector cells obtained from the subject compared to a reference population of immune effector cells.

23. In the subject - rescue or reverse immune effector cell (e.g., CAR T cell) exhaustion; - Increase immune effector cell activity or cytotoxicity of exhausted immune effector cells (e.g., CAR T cells), or - Prevent, inhibit, reduce, or delay the development of immune effector cell (e.g., CAR T cell) exhaustion 1. Use of a bispecific polypeptide in the manufacture of a medicament for Preferably, the use wherein the subject has undergone or is undergoing treatment with engineered immune effector cells (e.g., CAR T cells).

24. 24. The method of any one of claims 1 to 23, for the treatment of a disease or condition selected from cancer, chronic infection (e.g., chronic bacterial or chronic viral infection), chronic inflammation or autoimmunity.

25. 25. The method of any one of claims 1 to 24, wherein the bispecific polypeptide binds to a professional APC that is an endogenous APC of the subject in need of treatment.

26. 26. The method of any one of claims 1 to 25, wherein the bispecific polypeptide binds to an APC selected from a dendritic cell, a macrophage and a B cell, more preferably a dendritic cell (DC).

27. 27. The method of any one of claims 1 to 26, wherein the bispecific polypeptide binds to an antigen of a professional APC selected from MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, CD206 and CD83.

28. 28. The method of any one of claims 1 to 27, wherein the bispecific polypeptide binds to an antigen on the CAR portion of the CAR T cell.

29. 29. The method of any one of claims 1 to 28, wherein the bispecific polypeptide binds to the antigen binding protein of the CAR, the hinge region of the CAR, a tag on the CAR (e.g., Myc, Flag, His, HA, SBP, GST, MBP, GFP, S, Strep, eXact or other tag), or any other region of the extracellular portion of the CAR.

30. 29. The method of any one of claims 1 to 28, wherein the bispecific polypeptide binds to an antigen on an immune effector cell that is not an antigen on a heterologous receptor such as a CAR.

31. 31. The method of claim 30, wherein the bispecific polypeptide binds to an antigen on the surface of an immune effector cell selected from CD3, CD4, CD8, CD25, CD127, CD196 (CCR6), CD27, CD28, CD45RA, CD45RO, CD62L, CD197, and HLA-DR.

32. 31. The method of claim 30, wherein the bispecific polypeptide binds to a heterologous tag on an immune effector cell.

33. 33. The method of any one of claims 1 to 32, wherein the bispecific polypeptide is in the form of a fusion protein, optionally wherein the first and second antigen-binding proteins are linked directly or via a hinge or linker region. Suitable linkers are further described herein.

34. 34. The method of any one of claims 1 to 33, wherein the first and second antigen-binding proteins of the bispecific polypeptide are in the form of antibodies or antigen-binding fragments thereof.

35. 35. The method of claim 34, wherein the first antigen-binding protein is in the form of an antibody, such as an immunoglobulin G (IgG1, IgG2, IgG3 or IgG4).

36. 36. The method of claim 35, wherein the first antigen-binding protein is in the form of an antibody fragment (e.g., scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2).

37. 37. The method of claim 35 or 36, wherein the second antigen-binding protein is in the form of an antibody, such as an immunoglobulin G (IgG1, IgG2, IgG3 or IgG4).

38. 37. The method of claim 35 or 36, wherein the second antigen-binding protein is in the form of an antibody fragment (e.g., scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2).

39. 39. The method of any one of claims 1 to 38, wherein the second antigen-binding protein is linked to the first antigen-binding protein via the C- or N-terminus, or an internal region of the first antigen-binding protein, preferably the second antigen-binding protein is linked to the first antigen-binding protein via the C-terminus of the heavy chain of the first antigen-binding protein.

40. 40. The method of any one of claims 1 to 39, wherein the bispecific polypeptide comprises a first antigen-binding protein for binding to CD40.

41. 41. The method of any one of claims 1 to 40, wherein the bispecific polypeptide comprises a second antigen binding protein for binding to a tag on the CAR of the CAR T cell.

42. 42. The method of claim 41, wherein the second antigen binding protein is for binding to a FLAG tag.

43. 43. The method of any one of claims 1 to 42, wherein the bispecific polypeptide comprises a first antigen-binding protein for binding to CD40, wherein the first antigen-binding protein comprises antigen-binding domains CDRH1, CDRH2 and / or CDRH3 having a variable heavy domain as defined in SEQ ID NO: 1 and / or antigen-binding domains CDRL1, CDRL2 and / or CDRL3 having a variable light chain as defined in SEQ ID NO:

2.

44. the first antigen-binding protein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, Optionally, the linker may be a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues; the first antigen-binding protein (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 4, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 5; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 6, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 8; or (iii) a complementarity determining region (CDR) 1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; a VH comprising a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO:5; and (iv) a CDR1 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO: 6; a CDR2 comprising, or consisting of, a sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 8; 44. The method of any one of claims 1 to 43, comprising an antigen-binding domain comprising:

45. 45. The method of any one of claims 1 to 44, wherein the bispecific polypeptide comprises a first antigen-binding protein comprising a heavy chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 1, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

46. 46. ​​The method of any one of claims 1 to 45, wherein the bispecific polypeptide comprises a first antigen-binding protein comprising a light chain variable domain comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO: 2, or a sequence which is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

47. 47. The method of any one of claims 1 to 46, wherein the bispecific polypeptide comprises a second antigen-binding protein for binding to the FLAG tag, and the second antigen-binding protein comprises CDRH1, CDRH2 and / or CDRH3 of an antigen-binding domain having a variable heavy chain defined in SEQ ID NO: 15 and / or CDRL1, CDRL2 and / or CDRL3 of an antigen-binding domain having a variable light chain defined in SEQ ID NO:

16.

48. the second antigen-binding protein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, Optionally, the linker may be a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues; the second antigen-binding domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 23, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 24, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 25; and (ii) a VL comprising a complementarity-determining region (CDR) 1 comprising or consisting of the sequence set forth in SEQ ID NO: 26, a CDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 27, and a CDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 28; or (iii) a complementarity determining region CDR1 comprising or consisting of an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 23, at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least a VH comprising a CDR2 comprising, or alternatively consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:25; a VH comprising a CDR3 comprising, or alternatively consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:25; (iv) a CDR1 comprising, or consisting of, an amino acid sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence of SEQ ID NO: 26; a VL comprising a CDR2 comprising, or consisting of, an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 22; and a CDR3 comprising, or consisting of, an amino acid sequence that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:

22.

48. The method of any one of claims 1 to 47, comprising an antigen-binding domain comprising:

49. 49. The method of any one of claims 1 to 48, wherein the bispecific polypeptide comprises a second antigen-binding protein comprising an antigen-binding domain comprising a variable heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

50. 50. The method of any one of claims 1 to 49, wherein the bispecific polypeptide comprises a second antigen-binding protein comprising an antigen-binding domain comprising a variable light chain comprising an amino acid sequence as set forth in SEQ ID NO: 16, or a sequence at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

51. 51. The method of any one of claims 1 to 50, wherein the bispecific polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 42 and 43.

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