Method for rescuing depleted immune cells

Abstract

The present invention relates to compositions and methods for preventing or rescuing the depletion of immune effector cells, in particular CAR T cells. The compositions and methods comprise bispecific polypeptides 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 the immune effector cell (e.g., a FLAG tag on a CAR T cell).

Description

Technical Field

[0001] The present disclosure generally relates to methods of reducing, preventing or rescuing exhausted immune cells expressing engineered receptors, such as chimeric antigen receptors or modified TCRs.

[0002] Related Applications

[0003] This application claims priority from Australian provisional application AU 2022902711, the entire contents of which are hereby incorporated by reference. Background Art

[0004] Immune cell exhaustion is an acquired state of immune cell dysfunction. Specifically, T cell exhaustion is an acquired state of T cell dysfunction and is a hallmark of cancer and chronic viral infection. Recently, treatments to reverse T cell exhaustion in cancer have been shown to be remarkably effective.

[0005] The use of engineered immune cells expressing genetically engineered antigen receptors, such as chimeric antigen receptor (CAR) T cells, has also been shown to be effective for the treatment of hematological malignancies. However, the development of CAR T cell depletion remains a significant obstacle to the wider application of this technology, particularly in the treatment of solid tumors.

[0006] T cell exhaustion (including CAR T exhaustion) is characterized by a progressive decline in effector function, usually manifested as a loss of T cell multifunctionality. Exhausted T cells are usually manifested by increased expression of inhibitory receptors (such as PD-1, LAG-3, and PD-L1), reduced production of effector cytokines, reduced proliferation rate, and reduced target cell killing activity. Regeneration of exhausted T cells can reactivate immunity.

[0007] Current strategies for regulating T cell exhaustion rely on directly regulating the expression of effector gene expression products such as immune checkpoints. Such regulation can produce undesirable side effects, as normal T cell function often requires such effector gene expression products to be at physiological levels. In addition, these strategies are susceptible to drug resistance and may cause immunopathology.

[0008] Therefore, there is a need to develop new compositions and methods for preventing or rescuing immune cell exhaustion, particularly T cell exhaustion.

[0009] The reference to any prior art in the specification is not an acknowledgement or implication that the prior art forms part of the common general knowledge in any jurisdiction, or that the prior art could reasonably be expected to be understood by a person skilled in the art, to be relevant and / or to be combined with other prior art. Summary of the invention

[0010] The present invention provides a method for rescuing immune effector cell activity or reversing immune effector cell exhaustion, the method comprising exposing immune effector cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide,

[0011] - 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 antigen binding protein that specifically binds to an antigen on said immune effector cell,

[0012] -optionally wherein the immune effector cell expresses a heterologous receptor comprising an antigen binding protein and a signaling protein,

[0013] Thereby rescuing immune effector cell activity or reversing immune effector cell exhaustion.

[0014] The present invention also provides a method for increasing the immune effector cell activity or cytotoxicity of exhausted immune effector cells, the method comprising exposing a plurality of immune effector cells exhibiting one or more signs of an exhausted phenotype to an effective amount of a bispecific polypeptide,

[0015] - 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 antigen binding protein that specifically binds to an antigen on said immune effector cell,

[0016] -optionally wherein the immune effector cell expresses a heterologous receptor comprising an antigen binding protein and a signaling protein,

[0017] Thereby increasing immune cell effector activity or cytotoxicity.

[0018] The present invention also provides a method for preventing, inhibiting, reducing the progression of immune effector cell exhaustion or delaying the onset of immune effector cell exhaustion, the method comprising exposing a plurality of immune effector cells having or suspected of having an exhausted phenotype to an effective amount of a bispecific polypeptide,

[0019] - 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 antigen binding protein that specifically binds to an antigen on said immune effector cell,

[0020] -optionally wherein the immune effector cell expresses a heterologous receptor comprising an antigen binding protein and a signaling protein,

[0021] Thereby preventing, inhibiting, reducing the progression of immune effector cell exhaustion or delaying the onset of immune effector cell exhaustion.

[0022] The present invention also provides a method for treating a disease or condition characterized by exhaustion of immune effector cells, the method comprising administering to a subject in need thereof an effective amount of a bispecific polypeptide,

[0023] - 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 antigen binding protein that specifically binds to an antigen on an immune effector cell of the subject,

[0024] Diseases or conditions characterized by depletion of immune effector cells are thereby treated.

[0025] In any embodiment, the immune effector cell is selected from a T cell, a NK cell, a NKT cell or a γδT cell. Preferably the effector immune cell is a T cell.

[0026] Preferably, the heterologous receptor expressed by immune effector cells is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) in conjunction with a target antigen (such as a tumor-associated antigen). It should be understood that in a preferred embodiment, the immune effector cells are recombinant immune effector cells expressing CAR or modified TCR, or immune effector cells derived therefrom.

[0027] Therefore, in a preferred embodiment, the immune effector cell is a T cell expressing CAR (CAR T cell). Optionally, the immune effector cell is a CAR NK cell or a CAR NKT cell.

[0028] In a particularly preferred embodiment, a method for rescuing or reversing CAR T cell exhaustion is provided, the method comprising exposing a plurality of CAR T cells to an effective amount of a bispecific polypeptide, optionally wherein the cells have or are suspected of having an exhausted phenotype,

[0029] - 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 antigen binding protein that specifically binds to an antigen on said CAR T cell,

[0030] Thereby rescuing or reversing CAR T cell exhaustion.

[0031] As used herein, rescuing or reversing immune cell effector exhaustion includes reversing at least one marker or sign of an exhausted phenotype in an immune effector cell.

[0032] In addition, a method for increasing CAR T cell activity or cytotoxicity is provided, the method comprising exposing a plurality of CAR T cells to an effective amount of a bispecific polypeptide, preferably wherein the cells have or are suspected of having an exhausted phenotype,

[0033] - 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 antigen binding protein that specifically binds to an antigen on said CAR T cell,

[0034] Thereby increasing CAR T cell activity or cytotoxicity.

[0035] In addition, a method for preventing, inhibiting, reducing the progression of CAR T cell exhaustion or delaying the onset of CAR T cell exhaustion is provided, the method comprising exposing a plurality of CAR T cells to an effective amount of a bispecific polypeptide, preferably wherein the cells have or are suspected of having an exhausted phenotype,

[0036] - 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 antigen binding protein that specifically binds to an antigen on said CAR T cell,

[0037] Thereby preventing, inhibiting, reducing or delaying the onset of CAR T exhaustion.

[0038] In any embodiment, exposing immune effector cells (preferably CAR T cells) to bispecific polypeptides includes contacting a population of immune effector cells (e.g., CAR T cells) obtained from a subject with an effective amount of bispecific polypeptides ex vivo. Optionally, the method includes further administering to the subject a step of immune effector cells contacted with bispecific polypeptides.

[0039] In any embodiment, exposing the immune cells (e.g., CAR T cells) to an effective amount of the bispecific polypeptide is performed in vivo. Thus, in such embodiments, the method comprises administering the bispecific polypeptide to a subject who has received or is receiving treatment with immune effector cells (e.g., CAR T cells), such that the invention provides the following methods: in a subject,

[0040] - Rescue of immune effector cell (e.g. CAR T cell) activity;

[0041] -Reversal of immune effector cell (e.g. CAR T cell) exhaustion;

[0042] - Increase immune effector cell (e.g. CAR T cell) activity or cytotoxicity; or

[0043] -Prevent, inhibit, reduce the progression of, or delay the onset of, immune effector cell (e.g., CAR T cell) exhaustion,

[0044] The method comprises administering to the 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 antigen binding protein that specifically binds to an antigen on the immune (e.g., CAR T) cell.

[0045] In a preferred embodiment, after the subject has been treated with CAR T cells (or other immune effector cell therapy), the bispecific polypeptide is applied to the subject. In such embodiments, preferably 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 applying CART cells (or other immune effector cell therapy) to the subject, the bispecific polypeptide is applied to the subject.

[0046] In any embodiment, upon administration of the bispecific polypeptide to a subject, an exhaustion phenotype of one or more immune effector cells, or a marker or parameter indicative of the exhaustion phenotype, is detectable or measurable in the subject or in a biological sample from the subject.

[0047] In any embodiment, upon administration of the bispecific polypeptide to a subject, an exhaustion phenotype of one or more immune effector cells, or a marker or parameter indicative of the exhaustion phenotype, has been detected or measured in the subject or in a biological sample from the subject.

[0048] Optionally, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, or at least or about 50% of the total immune effector cells (preferably CAR T cells) in a biological sample from the subject have an exhausted phenotype or at least one sign or marker of an exhausted phenotype.

[0049] In any embodiment, the methods of the invention further comprise determining the risk or likelihood that the subject has immune effector cells (such as CAR T cells) with an exhausted phenotype.

[0050] Therefore, the present invention also provides a method of treatment, which comprises:

[0051] - Provide or identify subjects who have received treatment with CAR T cells;

[0052] - Determination that the subject may have or is at risk for having CAR T cells with an exhausted phenotype;

[0053] - administering a bispecific polypeptide to said subject,

[0054] - 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 antigen binding protein that specifically binds to an antigen on said CAR T cell.

[0055] Determining whether a subject has or is at risk for having immune effector cells (such as CAR T cells) with an exhausted phenotype may 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.

[0056] In some embodiments, the exhaustion phenotype can include an increase in the level or extent of surface expression of a marker on an immune cell, or an increase in the percentage of immune cells that exhibit surface expression of one or more exhaustion markers, compared to a reference population of immune effector cells.

[0057] Examples of such genes or markers are further described herein and known to the skilled person.

[0058] In any embodiment, the exhaustion phenotype of a T cell or T cell population includes: an increase in the level or extent of surface expression on one or more T cells, or an increase in the percentage of T cells in the T cell population that exhibit 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 extent of activity exhibited by the T cell or T cell population after exposure to an antigen or antigen receptor-specific agent, compared to a reference T cell population under the same conditions.

[0059] 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 naive T cell population, a central memory T cell population, or a stem central memory T cell population. Optionally, the cells are from the same subject or the same species as the subject from which the immune effector cells with an exhausted phenotype are derived.

[0060] In some embodiments, reference immune effector cell group (a) is a subject matching group, including most of the immune effector cells separated from the blood of the subject from which the immune effector cells with exhaustion phenotype are derived, optionally wherein most of the immune effector cells do not express heterologous receptors (eg, CAR), and / or (b) obtained from the subject from which the immune effector cells with exhaustion phenotype are derived before receiving a certain dose of immune effector cells expressing heterologous receptors (eg, CAR). In other embodiments, reference immune effector cell group is a composition comprising a sample of immune effector cell therapy before being applied to a subject, or a pharmaceutical composition comprising immune effector cells expressing recombinant receptors, optionally wherein the composition is a cryopreserved sample.

[0061] The sample may be any biological sample comprising immune cells, such as a biological fluid, a tissue or a tumor. Preferably the biological sample is a whole blood sample or immune cells derived therefrom (such as a peripheral blood mononuclear cell PBMC sample).

[0062] The present invention also provides a use of a bispecific polypeptide in the manufacture of a medicament for: in a subject,

[0063] -Rescue or reverse exhaustion of immune effector cells (e.g., CAR T cells);

[0064] - Increase immune effector cell (e.g. CAR T cell) activity or cytotoxicity; or

[0065] -Prevent, inhibit, reduce the progression of, or delay the onset of, immune effector cell (e.g., CAR T cell) exhaustion,

[0066] Preferably wherein the subject has received or is receiving treatment with engineered immune effector cells (such as CART cells).

[0067] The present invention also provides a bispecific polypeptide or a pharmaceutical composition comprising the bispecific polypeptide, which is used for:

[0068] -Rescue or reverse exhaustion of immune effector cells (e.g., CAR T cells);

[0069] - Increase immune effector cell (e.g. CAR T cell) activity or cytotoxicity; or

[0070] -Prevent, inhibit, reduce the progression of, or delay the onset of immune effector cell (e.g., CAR T cell) exhaustion.

[0071] In any embodiment, the methods of the invention are used to rescue exhaustion of immune effector cells in the context of any disease or condition selected from: cancer, chronic infection (such as 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 such as cancer, chronic infection, chronic inflammation, or autoimmunity, comprising administering to a subject in need thereof a bispecific polypeptide as described herein.

[0072] 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 antigen binding protein that specifically binds to an antigen on a CAR T cell may be additionally referred to herein as a "BEAT".

[0073] Preferably, the professional APC is an endogenous APC of the subject in need of treatment. Preferably, the professional APC is selected from dendritic cells, macrophages and B cells, more preferably, dendritic cells (DC). Therefore, in a preferred embodiment, the first antigen binding protein of the bispecific polypeptide can bind to any antigen of the professional APC, for example, any antigen selected from the following: MHCII, Clec9a, PD-L1, PD-L2, galectin, CD11c, CD19, CD40, CD206 and CD83.

[0074] 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 can specifically bind to the antigen binding protein of the CAR, the hinge region of the CAR, a tag on the CAR (such as Myc, Flag, His, HA, SBP, GST, MBP, GFP, S, Strep, eXact or other tags) or any other region of the extracellular portion of the CAR.

[0075] Alternatively, the second antigen-binding protein of the bispecific polypeptide can specifically bind to an antigen on an immune effector cell (optionally wherein the immune effector cell expresses a heterologous receptor such as CAR). In such embodiments, the second antigen-binding protein of the bispecific polypeptide can specifically bind to an antigen on the surface of an immune effector cell (e.g., T cell), such as: CD3, CD4, CD8, CD25, CD127, CD196 (CCR6), CD27, CD28, CD45RA, CD45RO, CD62L, CD197, and HLA-DR). In other embodiments, immune effector cells can be engineered to express heterologous antigens or labels in addition to expressing CAR receptors, and the second antigen-binding protein of the bispecific polypeptide can bind to the heterologous antigen or label.

[0076] 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 region or a linker region. Suitable linkers are further described herein.

[0077] The first and second antigen-binding proteins of the bispecific polypeptide are preferably in the form of an antibody or its antigen-binding fragment. In any embodiment, the first antigen-binding protein and / or the second antigen-binding protein are in the form of an antibody such as immunoglobulin G (IgG1, IgG2, IgG3 or IgG4). Optionally, the first antigen-binding protein is in the form of an antibody such as immunoglobulin G (IgG1, IgG2, IgG3 or IgG4), and the second antigen-binding protein is in the form of an antibody fragment (such as scFv, dimeric scFv (di-scFv), Fab, Fv, F (ab') 2), wherein the second antigen-binding protein is connected to the first antigen-binding protein via the C-terminus or N-terminus or internal region of the first antigen-binding protein, preferably wherein the second antigen-binding protein is connected to the first antigen-binding protein 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 such as immunoglobulin G (IgG1, IgG2, IgG3 or IgG4), and the first antigen binding protein is in the form of an antibody fragment (such as scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2), wherein the first antigen binding protein is linked to the second antigen binding protein via the C-terminus or N-terminus or an internal region of the second antigen binding protein, preferably wherein 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.

[0078] In any 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.

[0079] In any 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.

[0080] In any embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding CD40 and a second antigen binding protein for binding a FLAG tag.

[0081] Preferably, the antigen binding protein for binding to CD40 is an activator of CD40.

[0082] In any embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding CD206 and a second antigen binding protein for binding a FLAG tag.

[0083] In any embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD40, and wherein the first antigen binding protein comprises:

[0084] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4

[0085] and FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0086] The sequence of any one of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1.

[0087] In any embodiment, the first antigen binding protein competitively inhibits binding of an antibody or antigen binding fragment thereof to CD40, wherein the antibody or antigen binding fragment thereof comprises a VH and a VL, wherein the VH comprises the sequence set forth in SEQ ID NO: 1 and the VL comprises the sequence set forth in SEQ ID NO: 2.

[0088] In any embodiment the first antigen binding protein comprises a CDRH1 , CDRH2 and / or CDRH3 having an antigen binding domain with a variable heavy domain as defined in SEQ ID NO:1.

[0089] In any embodiment the first antigen binding protein comprises CDRL1, CDRL2 and / or CDRL3 having an antigen binding domain of a variable light chain as defined in SEQ ID NO:2.

[0090] In any embodiment the first antigen binding protein comprises CDR1 , CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined by SEQ ID NO: 1 and a variable light chain as defined by SEQ ID NO:2.

[0091] Preferably, the first antigen binding protein comprises:

[0092] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0093] The linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0094] Optionally, the first antigen binding protein comprises:

[0095] (i) VH, wherein the VH comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 3, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 4, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 5, and

[0096] (ii) VL, wherein the VL comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 6, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 7, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 8;

[0097] or

[0098] (iii) a VH comprising a complementarity determining region (CDR) 1, a CDR2, and a CDR3, wherein the CDR1 comprises 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 of SEQ ID NO:3, or consists thereof, and the CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence of SEQ ID NO:4, or consists thereof; and the CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence of SEQ ID NO:5. NO:5 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 or consisting of the sequence shown in NO:5, and

[0099] (iv) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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 of SEQ ID NO:6, and the CDR2 comprises 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 of SEQ ID NO:6 NO:7 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 shown in NO:7, or consists of a sequence thereof, and the CDR3 comprises 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 shown in SEQ ID NO:8 or consists of a sequence thereof.

[0100] In any embodiment, the first antigen binding protein comprises a heavy chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1, or 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 thereto.

[0101] In any embodiment, the first antigen binding protein comprises a light chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or 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 thereto.

[0102] In any embodiment, the second antigen binding protein is used to bind to the FLAG tag, and the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises:

[0103] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4 and

[0104] FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0105] in:

[0106] FR1, FR2, FR3 and FR4 are each a framework region;

[0107] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0108] FR1a, FR2a, FR3a and FR4a are each framework regions;

[0109] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0110] The sequence of any one of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1.

[0111] In any 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., 2×FLAG, 3×FLAG, etc.).

[0112] In any embodiment, the second antigen binding protein is used to specifically bind 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 wherein the additional amino acids in the FLAG tag are hydrophilic amino acids (e.g., R (Arg), D (Asp), E (Glu) and K (Lys)) and / or amino acids with aromatic side chains (e.g., Y (Tyr), F (Phe), H (His) and W (Trp)).

[0113] In a preferred embodiment, the second antigen binding protein is capable of specifically binding to a FLAG-tag that contains, comprises or consists of the following 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 modified by amino acid insertions, deletions or substitutions, such as those derived from the above-mentioned FLAG-tags, in particular tags having the sequence DYKDDDDK.

[0114] In any embodiment, the FLAG tag is present at the N-terminus, C-terminus, or within the protein (e.g., CAR) to which the bispecific polypeptide is capable of binding, binds, or specifically binds.

[0115] In any embodiment, the second antigen binding protein competitively inhibits binding of an antibody to a FLAG tag, wherein the antibody comprises a VH and a VL, wherein the VH comprises the sequence shown in SEQ ID NO:15 and the VL comprises the sequence shown in SEQ ID NO:16.

[0116] In any embodiment, the second antigen binding protein comprises a CDRH1, CDRH2 and / or CDRH3 having an antigen binding domain of a variable heavy chain as defined in SEQ ID NO:15.

[0117] In any embodiment, the second antigen binding protein comprises CDRL1, CDRL2 and / or CDRL3 having an antigen binding domain of a variable light chain as defined by SEQ ID NO:16.

[0118] In any embodiment, the second antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined by SEQ ID NO:15 and a variable light chain as defined by SEQ ID NO:16.

[0119] In any embodiment the second antigen binding protein comprises:

[0120] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0121] wherein the sequence of any of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1, and optionally wherein the linker may be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0122] In any embodiment, the second antigen binding domain comprises an antigen binding domain comprising:

[0123] VH, wherein the VH comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists 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; the CDR2 comprises 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; NO:24 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:24; the CDR3 comprises or consists 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:25, and

[0124] VL, the VL comprises CDR1, CDR2, and CDR3, the 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; the CDR2 comprises 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 NO:27 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, or consists 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:22, or consists 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:22.

[0125] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising:

[0126] VH, the VH comprising CDR1, CDR2, and CDR3, the CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; the CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24; the CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25, and

[0127] VL, wherein the VL comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26; the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27; and the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 22.

[0128] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain comprising the amino acid sequence shown 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.

[0129] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising a variable light chain comprising the amino acid sequence shown 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.

[0130] In any embodiment of any aspect of the invention, the bispecific polypeptide comprises, consists essentially of or consists of the amino acid sequences of SEQ ID NOs: 42 and 43.

[0131] As used herein, the term "comprise" or variations of the term, such as "comprising" and "including", are not intended to exclude additional additives, components, integers or steps, unless the context requires otherwise.

[0132] Further aspects of the invention and further embodiments of the various aspects described in the preceding paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 Schematic diagram of the experimental protocol for stimulating CAR T cells with mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs) and restimulating with tumor cells or MoDCs.

[0134] Figure 2 Activation and exhaustion of CAR T cells 6 days after stimulation of CAR T cells with high-expressing mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs) and subsequent restimulation with tumor cells or MoDCs.

[0135] Figure 3CAR T cell phenotype after stimulation of CART cells with high-expressing mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs) and subsequent restimulation with tumor cells or MoDCs.

[0136] Figure 4 Schematic diagram of the experimental protocol for sorting pre-stimulated CAR T cells and then re-culturing the CAR T cells with mesothelin-positive tumor cells, mesothelin-negative tumor cells, monocyte-derived DCs (MoDCs), or a combination of monocyte-derived DCs (MoDCs) and BEAT for further stimulation and assessment of proliferation and phenotype.

[0137] Figure 5 T cell phenotypes of CAR T cells pre-treated with BEAT and MoDC (A) or tumor (B) pre-treated with BEAT and MoDC restimulated with BEAT and MoDC, negative tumor or positive tumor. CAR T cells pre-treated with BEAT and MoDC had higher central memory (TCM) and stem cell memory (TSCM) phenotypes compared to tumor pre-treated CAR T cells with higher effector memory phenotype (TEM).

[0138] Figure 6 T cell proliferation when CAR T cells were pre-stimulated with tumor (A) or BEAT and MoDC (B). CAR T cells pre-stimulated with BEAT and MoDC had higher proliferation compared to CAR pre-treated with tumor.

[0139] Sequence Description

[0140] Table 1. Exemplary sequences

[0141]

[0142]

[0143]

[0144] DETAILED DESCRIPTION

[0145] It should be understood that the invention disclosed and defined in this specification includes all optional combinations of two or more individual features mentioned in the text or drawings or clearly seen from the text or drawings. All these different combinations constitute various optional aspects of the present invention.

[0146] Throughout the specification, reference to a single step, composition of matter, group of steps, or group of compositions of matter shall encompass both one and multiple (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter, unless specifically stated otherwise or the context requires otherwise. 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, reference to "a" includes the single as well as two or more; reference to "an" includes the single as well as two or more; reference to "the" includes the single as well as two or more, and so forth.

[0147] Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications in addition to those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification individually or collectively, as well as any and all combinations or any two or more of the steps or features.

[0148] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0149] 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 and explained throughout the literature in sources such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991); DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996); FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and JE Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0150] Reference will now be made in detail to certain embodiments of the present invention. Although the present invention will be described in conjunction with the embodiments, it should be understood that its purpose is not to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all substitutions, modifications and equivalents that may be included within the scope of the invention as defined by the claims.

[0151] The inventors have surprisingly shown that bispecific polypeptides as described herein for binding to professional APCs and immune effector cells (such as CAR T cells) rescue the exhaustion phenotype and promote activation and expansion of exhausted immune cells in vivo. Therefore, the methods of the present invention are, in part, directed to methods for improving the efficacy of immune cell therapies such as CAR T cell therapies.

[0152] definition

[0153] The term "protein" should be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. Proteins may contain one or more unnatural amino acids.

[0154] The term "polypeptide" or "polypeptide chain" will be understood from the above paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0155] Amino acids may be referred to herein by either their commonly used full names (e.g., cysteine), by their commonly known three letter symbols (e.g., Cys), or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (e.g., C. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0156] As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, i.e., two heavy chains (H) and two light chains (L). Antigen-binding fragments, mutants, variants and derivatives thereof that retain the basic epitope binding characteristics of antibody molecules are also disclosed herein. Such mutants, variants and derivatives will be known to those skilled in the art, and illustrative examples thereof are described elsewhere herein.

[0157] The heavy chain of an antibody will typically comprise 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. The light chain will typically comprise a light chain variable region (LCVR or VL) and a light chain constant region, CL. The VH region and the VL region can be further subdivided into hypervariable regions, also known as complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL typically comprises three CDs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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.

[0158] As used herein, the term "antigen-binding fragment" or "antibody fragment" means one or more fragments of an antibody that retains the ability to specifically bind to a target antigen. Illustrative examples of antigen-binding fragments include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) single-chain variable fragments (scFv) consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al. 1989, Nature, 341: 544-6), which contain a single variable domain; and (vi) isolated CDRs.

[0159] As used herein, "variable region" refers to the portion of the light chain and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen, and comprises 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 comprises three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. H V refers to the variable region of the heavy chain. L Refers to the variable region of the light chain.

[0160] The description and definition of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be as discussed 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) 45:3832-9; or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997; or further illustrated by the IMGT system discussed in Giudicelli et al., Nucleic Acids Res.25:206-211 1997.

[0161] "Framework regions" (FRs) are those 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, wherein FR H1 corresponds to V H FR1, FR H2 corresponds to V H FR 2, FR H3 corresponds to V H FR 3, and FR H4 corresponds to V H FR 4. Similarly, V L The FRs of V L1, V L2, V L3 and V L4 are referred to herein as FR L1, FR L2, FR L3 and FR L4, where FR L1 corresponds to V L FR1, FR L2 corresponds to V L FR 2, FR L3 corresponds to V L FR3 and FRL4 correspond to V L FR 4.

[0162] As used herein, the term "Fv" should be understood to mean any protein, whether comprising multiple polypeptides or a single polypeptide, in which V L and V H The V that forms the antigen-binding domain H and V L Can be located in a single polypeptide chain or in different polypeptide chains. In addition, the Fv of the present invention (and any protein of the present invention) can have multiple antigen binding domains that may or may not bind to the same antigen. The term should be understood to cover fragments directly derived from antibodies as well as proteins corresponding to such fragments produced using recombinant methods. In some examples, V H Not associated with the heavy chain constant domain (C H )1 Connect and / or V L Not associated with the light chain constant domain (C L ). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher order complexes, or any of the foregoing linked to their constant regions or domains, such as CH2 or CH3 domains, e.g., minibodies.

[0163] "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by digesting whole antibodies with papain to produce fragments consisting of an intact light chain and a portion of a heavy chain, or can be produced using recombinant methods. "Fab' fragments" of antibodies can be obtained by treating whole antibodies with pepsin followed by reduction to produce a fragment consisting of an intact light chain and a portion of a heavy chain. HA molecule consisting of a portion of a heavy chain containing a single constant domain. Two Fab' fragments are obtained for each antibody treated in this way. Fab' fragments can also be produced by recombinant methods. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked together by two disulfide bonds, and is obtained by treating the entire antibody molecule with pepsin but without subsequent reduction. The "Fab2" fragment is a recombinant fragment comprising two Fab fragments connected using, for example, a leucine zipper or a CH3 domain. "Single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv), 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.

[0164] As used herein, the term "antigen" refers to a molecule bound by an "antibody," "antibody fragment," or "bispecific polypeptide." An antigen may be a protein recognized by an immunoglobulin, in which case the site on the protein bound by the immunoglobulin is called an "epitope."

[0165] As used herein, the term "binding" with respect to the interaction of an antigen binding protein or its antigen binding domain with an antigen means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than a general protein. If an antibody binds to epitope "A", then in a reaction containing labeled "A" and a protein, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0166] As used herein, the term "specific binding" should be understood to mean that the reaction or association of the antigen-binding proteins of the present invention with a specific antigen or a cell expressing the specific antigen is more frequent, faster, longer lasting and / or has a higher affinity than the reaction or association with an alternative antigen or cell. For example, the antigen-binding protein binds to an antigen with an affinity much higher than it to other related molecules (e.g., 1.5 times or 2 times or 5 times or 10 times or 20 times or 40 times or 60 times or 80 times to 100 times or 150 times or 200 times). In an example of the present invention, the antigen-binding protein "specifically binds" an antigen with an affinity at least 1.5 times or 2 times or higher (e.g., 5 times or 10 times or 20 times or 50 times or 100 times or 200 times) than it to any other antigen. Usually, but not necessarily, reference to binding means specific binding, and each term should be understood to provide clear support for another term.

[0167] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. These terms mean containing and / or expressing exogenous genes or nucleic acid sequences, which in turn modify the genotype or phenotype of cells or their progeny. In particular, these terms refer to the fact that cells, preferentially immune cells, can be operated by recombinant methods known in the art to stably or transiently express peptides or proteins that are not expressed in these cells in a natural state. For example, immune cells are engineered to express artificial constructs such as chimeric antigen receptors on their cell surfaces. For example, CAR sequences can be delivered to cells using adenovirus, based on adeno-associated virus (AAV), retrovirus or lentiviral vectors or any other pseudotype 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).

[0168] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide comprising an antigen binding domain connected to at least one intracellular signaling domain. The antigen binding domain of CAR is a functional portion of CAR that specifically binds (i.e., specifically targets) an antigen expressed on a cancer cell (i.e., "tumor-associated antigen"). Examples of tumor-associated antigens are known to those skilled in the art, and illustrative examples thereof include Her2, CEA, FBP, CD19, and BCMA.

[0169] 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 a tumor-associated antigen is not expressed by non-tumor cells (i.e., when it is unique to tumor cells), it may also be referred to as a "tumor-specific antigen". When a tumor-associated antigen is not unique to tumor cells, it is also expressed on non-tumor cells under conditions where an immune tolerance state to the antigen cannot be induced. The expression of an antigen on a tumor can occur under conditions that enable the immune system to respond to the antigen. Tumor-associated antigens can be antigens expressed on non-tumor cells during fetal development when the immune system is immature and cannot respond, or they can be antigens that are usually present at low levels on normal cells but expressed at much higher levels on tumor cells. Compared with corresponding non-tumor tissues, those tumor-associated antigens of greatest clinical interest are differentially expressed, and allow specific T cells or immunoglobulins to preferentially recognize tumor cells.

[0170] As used herein, the term "cancer" means any condition associated with abnormal cell proliferation. Such conditions will be known to those skilled 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 breast cancer, pancreatic cancer and lung cancer.

[0171] As used herein, the term "treatment" refers to any and all uses of remedying a condition or symptom in any way, or otherwise preventing, hindering, delaying, eliminating or reversing the onset or progression of cancer or other undesirable symptoms. Therefore, the term "treatment" and the like are considered to be in their broadest possible context. For example, treatment does not necessarily mean that the subject is treated until full recovery or cure. In a condition exhibiting or characterized by a variety of symptoms, treatment does not necessarily remedy, prevent, hinder, delay, eliminate or reverse all of the symptoms, but may remedy, prevent, hinder, delay, eliminate or reverse one or more of the symptoms.

[0172] The term "immune cell" or "immune effector cell" refers to a cell that can be a part of the immune system, i.e., adaptability (i.e., cell or fluid) or the innate immune system and performs a specific effector function, such as α-βT cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphocytes (ILC), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-δT cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages, or any hematopoietic progenitor cells such as pluripotent stem cells and early progenitor cell subgroups that can mature or differentiate into somatic cells. Cells can be naturally occurring or produced by cytokine exposure, artificial / genetically modified cells (such as iPSC and other artificial cell types). Immune cells can be artificial cell subgroups, including induced pluripotent stem cells and cells matured therefrom. Preferred immune cells are cells with cytotoxic effector functions, such as α-βT cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells or γ-δT cells. "Effector function" means a specialized function of a cell, for example, in T cells, the effector function may be cytolytic activity or helper cell activity, including the secretion of cytokines.

[0173] Treatment

[0174] The present invention relates to methods for rescuing or reversing immune effector cell exhaustion, increasing immune effector cell activity or potency (particularly in the context of exhaustion), and preventing, inhibiting, reducing the progression of immune effector cell exhaustion or delaying the onset of immune effector cell exhaustion.

[0175] As described elsewhere herein, the immune effector cells to be prevented or rescued from exhaustion can be any immune effector cells, illustrative examples of which include T cells, tumor infiltrating lymphocytes (TIL) natural killer (NK) cells, natural killer T cells (NKT cells) and γ-δ cells (γδ cells). T cells can be cytotoxic T cells (CTL; ​​CD8+T cells), CD4+cos+T cells, CD4-cos-T cells or any other subpopulation of T cells. Other illustrative examples of suitable T cells include T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD2S, CD45RA, CD45RO, CD62L, CD127, CD197 and HLA-DR.

[0176] In a preferred embodiment, the immune cell is an immune cell expressing a heterologous chimeric antigen receptor (CAR) or a modified T cell receptor (TCR). Such receptors and their use in treating various disorders and diseases, especially cancer, are well known to those skilled in the art.

[0177] The sources of cells used in the methods according to the invention are known to those skilled in the art, and illustrative examples thereof include peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment, the cells are derived from whole blood.

[0178] In any embodiment, the immune cells are derived from autologous cells. In another embodiment, the immune cells are derived from allogeneic cells.

[0179] The term "autologous" refers to any material derived from the same individual into which it is to be reintroduced.

[0180] The term "allogeneic" refers to any material derived from a different individual of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species can be sufficiently different genetically to interact antigenically.

[0181] It will be appreciated that in preferred embodiments of the invention, these methods involve rescuing the exhaustion of CAR T cells that have already been administered to a subject and require reactivation.

[0182] Administration of CAR-expressing immune cells and bispecific polypeptides or pharmaceutical compositions as described herein can be achieved by formulating the immune cells and bispecific polypeptides or pharmaceutical compositions in the same composition (e.g., for simultaneous co-administration), or they can be formulated as different compositions for sequential administration.

[0183] It will be appreciated that sequential administration may be preferred given the focus of the methods of the invention on rescuing immune cell exhaustion, activating immune cells that display an exhausted phenotype, or preventing or delaying immune cell exhaustion.

[0184] "Sequentially" administration means that there is an interval between the administration of immune cells and bispecific polypeptides or pharmaceutical compositions comprising the bispecific polypeptides. The interval between sequential administration can be seconds, minutes, hours or days. Preferably, 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 longer are separated between the initial administration of immune cells (eg, CAR T cells) and bispecific polypeptides.

[0185] In one embodiment, periodic re-administration of the bispecific polypeptide may be required to achieve the desired therapeutic effect.

[0186] In some embodiments, the administration of the bispecific polypeptide is performed in a cyclical regimen involving the administration of an effective amount of the bispecific polypeptide in the following pattern: (i) daily administration for a period of more than one week, (ii) daily administration, but no more than 6 days per week, for a period of more than one week, (ii) daily administration, but no more than 5 days per week, for a period of more than one week; or daily administration, but no more than 4 days per week, for a period of more than one week. In certain embodiments, the administration of the bispecific polypeptide is performed in a cyclical regimen involving the administration of an effective amount of the bispecific polypeptide every day, no more than 5 days per week, for a period of more than one week.

[0187] In some embodiments of any method provided herein, the bispecific polypeptide is administered at or after the peak or maximum level of cells of T cell therapy can be detected in the blood of the subject. In some embodiments, the bispecific polypeptide is administered from about 14 days to about 35 days after the start of T cell therapy. In certain embodiments, the bispecific polypeptide is administered from about 21 days to about 35 days after the start of T cell therapy. In other embodiments, the bispecific polypeptide is administered from about 21 days to about 28 days after the start of T cell therapy. In some embodiments, the bispecific polypeptide is administered on or about the 21st day, on or about the 22nd day, on or about the 23rd day, on or about the 24th day, on or about the 25th day, on or about the 26th day, on or about the 27th day, or on or about the 28th day after the start of T cell therapy. In certain embodiments, the bispecific polypeptide is administered on or about the 28th day after the start of T cell therapy. In some embodiments of any method provided herein, after the administration of T cell therapy, when the bispecific polypeptide is administered, the subject does not show severe toxicity.

[0188] In some embodiments of any method provided herein, bispecific polypeptides are administered on or about the 28th day after T cell therapy is started. In some embodiments of any method provided herein, after T cell therapy is administered, when bispecific polypeptides are started, the subject does not show severe toxicity. In some embodiments, severe toxicity is severe cytokine release syndrome (CRS), optionally grade 3 or higher, long-term grade 3 or higher, or grade 4 or grade 5 CRS; and / or severe toxicity is severe neurotoxicity, optionally grade 3 or higher, long-term grade 3 or higher, or grade 4 or grade 5 neurotoxicity.

[0189] In some embodiments of any method provided herein, if the subject exhibits toxicity, optionally hematological toxicity, after administration of the bispecific polypeptide, the administration of the bispecific polypeptide is suspended and / or the cycle regimen is modified. In some embodiments, toxicity is selected from severe neutropenia, optionally febrile neutropenia, long-term grade 3 or higher neutropenia. In some embodiments, the administration of the bispecific polypeptide is restarted after the subject no longer exhibits toxicity. In some embodiments, after the administration of the bispecific polypeptide is restarted, the cycle regimen is modified. In some embodiments, modifying the cycle regimen involves reducing the amount of bispecific polypeptide administered and / or reducing the frequency of administration of the bispecific polypeptide. In certain embodiments, modifying the cycle regimen involves reducing the amount of bispecific polypeptide administered.

[0190] The method of the present invention also encompasses the in vitro contact of immune cells with bispecific polypeptides.For example, in the case where the patient receives treatment with immune cells (such as CAR T cells), and those cells are currently showing an exhaustion phenotype, cells can be taken out from the patient, and contacted with the bispecific polypeptide in vitro to save the exhaustion phenotype and reactivate the immune cells.In such embodiments, immune cells can also be subjected to a combination therapy with a bispecific polypeptide as described herein and another known immune cell activator.The example of such immune cell activation includes providing a primary stimulation signal by, for example, a T cell TCR / CD3 complex or via stimulation of CD2 surface proteins, and providing a secondary costimulatory signal by auxiliary molecules such as CD28 or 4-1BBL.In addition to providing a primary stimulation signal by a TCR / CD3 complex or via CD2, the induction of T cell responses requires a second costimulatory signal.In a specific embodiment, a CD28 binding agent can be used to provide a costimulatory signal. Suitable co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible co-stimulatory 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 to B7-H3.

[0191] The subject in need of treatment can be a human or a mammal of great economic and / or social importance to humans, for example, carnivores other than humans (e.g., cats and dogs), pigs (e.g., domestic pigs, hogs and wild boars), ruminants (e.g., cattle, bulls, sheep, giraffes, deer, goats, bison and camels), horses and birds (including endangered birds, those species kept in zoos) and poultry (more particularly, domesticated poultry, e.g., poultry such as turkeys, chickens, ducks, geese, guinea fowls, etc.), because they are of great economic importance to humans. The term "subject" does not indicate a particular age. Therefore, it is intended to cover adult, adolescent and neonatal subjects.

[0192] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to any subject to which the present disclosure may be applied. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0193] As used herein, the term "therapeutically effective amount" means that when administered to a mammal, particularly a human, in need of such treatment, the amount of the bispecific polypeptide is sufficient to rescue or prevent the exhaustion phenotype in immune effector cells (such as CAR T cells). The exact amount of the bispecific polypeptide to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the subject.

[0194] In any embodiment, the methods provided can enhance T cell therapy, such as CAR-T cell therapy, which can improve the outcome of treatment in some aspects. In some embodiments, these methods are particularly advantageous in the following subjects: subjects in which the cells of the T cell therapy show weak expansion, have been exhausted, show reduced or reduced persistence, and / or have cancers that are resistant or refractory to other therapies, aggressive or high-risk cancers, and / or subjects who show or are likely to show a relatively low response rate to CAR-T cell therapy without the administration of a bispecific polypeptide compared to another type of cancer or compared to the administration of a different CAR-T cell therapy.

[0195] In some embodiments, the method provided is used when T cell therapy (e.g., CAR T cells) can show or may show exhaustion characteristics. In some embodiments, after T cells have reached peak amplification and the number in the subject's blood begins to decline, the exhaustion phenotype is obvious. In some embodiments, compared to the time (baseline) before T cells are about to be exposed to antigens or the time point when cells have been exposed to antigens but continue to proliferate and have not yet reached peak amplification, the T cells (CAR T cells) of T cell therapy are exposed to or contacted with a bispecific polypeptide as described herein when T cells show an increase in hypofunction or exhaustion. In some embodiments, compared to a previous earlier time point, the increase in hypofunction or exhaustion can be determined by the increase in the expression of exhaustion markers. In some embodiments, the increase in hypofunction or exhaustion state, such as the increase in the expression of exhaustion markers, is the time after T cell therapy (e.g., CAR T cells) is administered to a subject suffering from a disease or condition related to an antigen targeted by T cell therapy. Following administration to a subject, T cells, e.g., T cells in peripheral blood, can be monitored for markers of T cell activation or exhaustion such as PD-1, TIM-3, and LAG-3, or as further described herein.

[0196] Methods for determining the exhaustion phenotype of immune effector cells are known to those skilled in the art and include determining the expression of various genes by immune cells, or determining the presence or absence of specific cell surface markers. In other words, exhaustion markers and methods for assessing / determining exhaustion markers in immune cell samples should be familiar to those skilled in the art.

[0197] In some embodiments of any method provided herein, the exhaustion phenotype of a T cell or T cell population includes: an increase in the level or extent of surface expression on one or more T cells, or an increase in the percentage of T cells in the T cell population that expresses surface expression of one or more exhaustion markers, optionally 2, 3, 4, 5 or 6 kinds of exhaustion markers, compared to a reference T cell population under the same conditions; or a decrease in the level or extent of activity exhibited by the T cell or T cell population after exposure to an antigen or antigen receptor-specific agent, compared to a reference T cell population under the same conditions. In some embodiments, the increase in level, extent or percentage is greater than or equal to or about 1.2 times, equal to or about 1.5 times, equal to or about 2.0 times, equal to or about 3 times, equal to or about 4 times, equal to or about 5 times, equal to or about 6 times, equal to or about 7 times, equal to or about 8 times, equal to or about 9 times, equal to or about 10 times or more. In other embodiments, the level, degree, or percentage is reduced by more than or about 1.2 times, by or about 1.5 times, by or about 2.0 times, by or about 3 times, by or about 4 times, by or about 5 times, by or about 6 times, by or about 7 times, by or about 8 times, by or about 9 times, by or about 10 times, or more.

[0198] In some embodiments of any of the methods provided herein, the reference T cell population is a T cell population known to have a non-exhausted phenotype, is a naive T cell population, is a central memory T cell population, or is a stem central memory T cell population, optionally from the same subject, or is of the same species as the subject from which the T cell or cells having the exhausted phenotype are derived.

[0199] In some embodiments of any of the methods provided herein, the reference T cell population (a) is a subject-matched population comprising a majority of T cells isolated from the blood of a subject from which a T cell or multiple T cells with an exhausted phenotype are derived, optionally wherein a majority of the T cells do not express the recombinant receptor, and / or (b) is obtained from a subject from which a T cell or multiple T cells with an exhausted phenotype are derived prior to receiving administration of a dose of T cells expressing the recombinant receptor. In some embodiments, the reference T cell population is a composition comprising a sample of a T cell therapy prior to administration to a subject, or a pharmaceutical composition comprising T cells expressing the recombinant receptor, optionally wherein the composition is a cryopreserved sample.

[0200] In some embodiments of any method provided herein, one or more exhaustion markers are inhibitory receptors. In some embodiments, one or more exhaustion markers are selected from PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA and TIGIT.

[0201] In some embodiments of any method provided herein, activity or proliferation, cytotoxicity or production of one or more of an inflammatory cytokine or a combination of inflammatory cytokines, optionally wherein one cytokine or a combination of cytokines is selected from IL-2, IFN-γ and TNF-α. Other markers of immune cell activation include CD25 and CD69. In some embodiments, exposure to the antigen or antigen receptor specific reagent includes incubation with an antigen or antigen receptor specific reagent, optionally a reagent that binds a recombinant receptor, wherein the antigen is optionally a target antigen. In some embodiments, the antigen or antigen receptor specific reagent includes a target cell expressing an antigen, optionally a cell of the disease, disorder or condition. In certain embodiments, the target antigen is a human antigen.

[0202] According to the methods of the present invention, it is also within the capabilities of the skilled person to be able to assess the efficacy of the treatment as described herein. In other words, the skilled person can easily determine whether immune cell depletion has been prevented, rescued, etc. by evaluating one or more depletion markers or evaluating one or more immune cell activation markers.

[0203] In some embodiments of any of the methods provided herein, administration of the bispecific polypeptide: reverses the exhausted phenotype of T cells expressing the recombinant receptor in the subject; prevents, inhibits, or delays the onset of the exhausted phenotype in T cells expressing the recombinant receptor in the subject; or reduces the level or extent of the exhausted phenotype in T cells expressing the recombinant receptor in the subject; or reduces the percentage of the total number of T cells expressing the recombinant receptor in the subject that have the exhausted phenotype.

[0204] In some embodiments of any of the methods provided herein, administration of the bispecific polypeptide is initiated after administration of the T cell therapy, and following administration of the bispecific polypeptide or initiation thereof, the subject exhibits recovery or rescue of antigen or tumor specific activity or function of T cells expressing the recombinant receptor in the subject, optionally wherein said recovery, rescue, and / or initiation of administration of the bispecific polypeptide is at a time point after the T cells expressing the recombinant receptor in the subject or in the blood of the subject have exhibited an exhausted phenotype.

[0205] In some embodiments of any of the methods provided herein, administration of the bispecific polypeptide comprises administration in an amount, frequency, and / or duration effective to: (a) achieve an increase in antigen-specific or antigen receptor-driven activity of naive or non-exhausted T cells (which optionally include T cells expressing said recombinant receptor) in a subject following exposure of the T cells to an antigen or antigen receptor-specific agent, compared to said administration in the absence of said bispecific polypeptide; or (b) prevent, inhibit, or delay the onset of an exhaustion phenotype in naive or non-exhausted T cells (which optionally include T cells expressing said recombinant receptor) in a subject following exposure of the T cells to an antigen or antigen receptor-specific agent, compared to said administration in the absence of said bispecific polypeptide; or (c) reverse the exhaustion phenotype of exhausted T cells (which optionally include T cells expressing said recombinant receptor) in a subject, compared to said administration in the absence of said subject.

[0206] In some embodiments, administration of the bispecific polypeptide comprises administration in an amount, frequency, and / or duration effective to achieve: (i) achieving an increase in said activity and (ii) preventing, inhibiting, or delaying said onset of said exhaustion phenotype and / or reversing said exhaustion phenotype. In some embodiments, the T cells in the subject comprise T cells expressing said recombinant receptor, and / or said antigen is a target antigen.

[0207] In some embodiments of any of the methods provided herein, the dose of the genetically engineered T cells for simultaneous or sequential administration comprises from equal to or about 1×10 5 Up to 5×10 8 Total CAR-expressing T cells, 1×10 6 Up to 2.5×10 8 Total CAR-expressing T cells, 5×10 6 Up to 1×10 8 Total CAR-expressing T cells, 1×10 7 Up to 2.5×10 8 Total CAR-expressing T cells, 5×10 7 Up to 1×10 8 In some embodiments of any of the methods provided herein, the dose of the genetically engineered T cells comprises at least equal to or at least about 1×10 5 CAR-expressing cells, at least equal to or at least about 2.5×10 5 CAR-expressing cells, at least equal to or at least about 5×10 5 CAR-expressing cells, at least equal to or at least about 1×10 6CAR-expressing cells, at least equal to or at least about 2.5×10 6 CAR-expressing cells, at least equal to or at least about 5×10 6 CAR-expressing cells, at least equal to or at least about 1×10 7 CAR-expressing cells, at least equal to or at least about 2.5×10 7 CAR-expressing cells, at least equal to or at least about 5×10 7 CAR-expressing cells, at least equal to or at least about 1×10 8 CAR-expressing cells, at least equal to or at least about 2.5×10 8 CAR-expressing cells or at least equal to or at least about 5×10 8 In certain embodiments, the dose of genetically engineered T cells comprises equal to or about 5×10 7 In other embodiments, the dose of genetically engineered T cells includes equal to or about 1×10 8 CAR-expressing cells.

[0208] In some embodiments of any of the methods provided herein, the dose of cells is administered parenterally, optionally intravenously.

[0209] 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.

[0210] In some embodiments of any of the methods provided herein, the dose of genetically engineered T cells includes CD4+ T cells expressing CAR and CD8+ T cells expressing CAR, and administration of the dose includes administration of multiple separate compositions, the multiple separate compositions including a first composition and a second composition, the first composition comprising one of CD4+ T cells and CD8+ T cells, the second composition comprising the other of CD4+ T cells or CD8+ T cells.

[0211] The dosage of the bispecific polypeptide for direct administration to a subject or for in vitro exposure to immune cells can be determined by those skilled in the art and will generally depend on factors including, but not limited to, the type, size, stage, and receptor status of the tumor, in addition to the age, weight, and general health of the subject. Another determining factor may be the risk of developing a recurrent disease. For example, a more aggressive treatment regimen may be used for subjects identified as being at high risk or at higher risk or developing a recurrent disease, compared to subjects who are considered to be at low risk or at lower risk of developing a recurrent disease. Similarly, a more aggressive treatment regimen may be used for subjects identified as having a more advanced cancer, such as stage III or IV disease, compared to subjects with mid- to late-stage cancer.

[0212] Bispecific peptides

[0213] As used herein, the term "bispecific polypeptide" refers to a polypeptide that can specifically bind to two different target antigens at the same time. The bispecific polypeptide used in the methods described herein comprises two structurally different 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.

[0214] Preferably, the binding protein comprises an antigen binding protein for binding to a target antigen on a professional APC, more preferably an endogenous professional APC, of ​​a subject in need of treatment. Examples of such antigens and such cells are further described herein.

[0215] Preferably, immune effector cells are immune effector cells expressing heterologous receptors such as chimeric antigen receptors (CAR), which include antigen binding domains and signal transduction domains. Therefore, in preferred embodiments, bispecific polypeptides include antigen binding domains for binding to antigens on immune cells expressing CAR (such as CAR T cells or CAR NK cells).

[0216] It will also be appreciated that bispecific polypeptides can be used to bind to a target antigen on an APC and a target antigen on a heterologous receptor (e.g., a CAR) expressed by an immune cell.

[0217] The antigen binding protein of the bispecific polypeptide used according to the present invention can comprise an antibody or an antigen binding fragment thereof. For example, the bispecific polypeptide can comprise the polypeptide sequence (i.e., domain) of one or more antibodies or antibody fragments. Examples of antibody fragments are further described herein.

[0218] In any embodiment, the 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.

[0219] The bispecific polypeptides used in the methods as described herein may be variously referred to as "bispecific engagers of APCs and T cells" or "BEATs".

[0220] As used herein, the term "antigen presenting cell" or "APC" preferably refers to professional antigen presenting cells (e.g., dendritic cells, macrophages, B cells, etc.) that are different from non-professional antigen presenting cells (e.g., fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells, vascular endothelial cells, etc.). In a preferred embodiment, APCs are professional antigen binding cells, preferably endogenous professional APCs, of a subject in need of treatment. In certain embodiments, endogenous professional APCs are not cells of a tumor or cancer being treated in a subject.

[0221] In any embodiment, the first antigen binding protein of the bispecific polypeptide specifically binds to an APC antigen selected from MHCII, Clec9a, PD-L1, PD-L2, galectins, CD11c, CD19, CD40, CD206 (also known as mannose receptor) and CD83. MHCII is expressed on dendritic cells (DC), mononuclear phagocytes, some endothelial cells, thymic epithelial cells and B cells; Clec9a is expressed on a small subset of BDCA+ dendritic cells and CD14+ / CD16- monocytes; PD-L1 and PD-L2 are expressed on macrophages, myeloid DC, B cells and vascular endothelial cells; galectins are expressed by T helper cells and B cells; CD11c is expressed at high levels by DC; CD19 is expressed on all professional B cells to mature B cells and follicular DC; CD40 is expressed by dendritic cells, B cells and macrophages; and CD83 is mainly expressed by mature DC. 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.

[0222] In any embodiment, the second antigen binding protein of the bispecific polypeptide specifically binds to an antigen on an immune effector cell expressing CAR or a similar receptor. For example, the second binding protein of the bispecific polypeptide used in the method of the present invention can bind to an antigen on an immune cell, wherein the antigen is not part of the CAR, but an antigen present on the cell surface of the immune cell. Alternatively, the second antigen binding protein can directly bind to the CAR expressed by the immune cell, such as an antigen binding protein binding to the CAR or an extracellular region of the CAR that does not participate in antigen binding, as further described below.

[0223] In certain embodiments, the CAR present on the immune cell may include additional amino acids or molecules for being bound by a second antigen binding protein. As known in the art, the CAR construct can be designed to include a "tag", which is generally a short amino acid sequence specifically recognized by an antibody. In some embodiments, the immune cell is a T cell or NK cell engineered to express a CAR comprising a tag. In the context of such embodiments, the second antigen binding protein of the bispecific polypeptide can bind to the tag, or it can bind to the region of the CAR other than the tag.

[0224] Illustrative examples of tags that may be present on CAR include peptide tags (e.g., FLAG-tags, HA-tags, His-tags, Myc-tags, S-tags, SBP-tags, Strep-tags, eXact-tags) and protein tags (e.g., GST-tags, MBP-tags, GFP-tags, tags in the form of proteins for binding antibodies or other proteins, nucleoproteins (such as leucine zippers) or any other protein modification to CAR). In any embodiment, the antigen or affinity tag on CAR is a c-Myc-tag or a FLAG-tag.

[0225] In some embodiments, the immune cell is a T cell or NK cell engineered to express a CAR that does not contain a tag. In some embodiments, the immune cell is a T cell or NK cell engineered to express a CAR that does not contain a tag or any heterologous tumor-associated antigen or a fragment of a tumor-associated antigen.

[0226] In certain embodiments, immune cells are engineered to express CAR T cells or NK cells, and bispecific polypeptides bind to the extracellular portion of CAR. The extracellular domain of CAR includes an antigen binding domain that is generally used to bind tumor-related or tumor-specific antigens. Typically, the antigen binding domain is an antibody fragment, such as Fab or scFv. Most typically, the antigen binding domain is scFv. The extracellular domain also generally includes a spacer (or hinge) region connecting the antigen binding domain to the transmembrane domain. The spacer region can be derived from immunoglobulins such as IgG1 or IgG4, or it can be derived from other cell surface proteins (including but not limited to CD4, CD8 or CD28).

[0227] Therefore, in certain embodiments, the second antigen binding protein of the bispecific polypeptide used in the method of the present invention can be used to bind to the antigen binding domain of the CAR. Therefore, the second antigen binding protein of the bispecific polypeptide can be derived from an anti-idiotypic antibody or an antigen binding fragment thereof, wherein the anti-idiotypic antibody is an anti-idiotypic antibody of the antibody portion of the CAR.

[0228] Anti-idiotypic antibodies are known in the art, and the skilled artisan will be fully capable of utilizing the antigen binding domains of these antibodies in the design of bispecific polypeptides for use in the methods of the invention. Thus, in some embodiments, the second antigen binding domain of the bispecific polypeptide used in the methods of the invention comprises an antibody or antibody fragment derived from an anti-idiotypic antibody specific for anti-CD 19, anti-Her2, anti-mesothelin or other antibodies, or an antigen binding fragment of an anti-idiotypic antibody for binding to an antibody present in the CAR.

[0229] Many anti-idiotypic antibodies are known in the art. For example, International Patent Application Publication No. WO 2014 / 190273 and Jena et al. 2013, PLoS One, 8 (3): e57838 describe an anti-idiotypic antibody (mAb clone number 136.20.1) recognizing anti-CD19 scFv FMC63, which is used in many CAR constructs currently being developed.

[0230] In certain embodiments, the second antigen-binding protein of the bispecific polypeptide used 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 that may have one or more of the same CDRs as mAb clone number 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 that may have one or more (e.g., two) variable regions from mAb clone number 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 number 136.20.1.

[0231] Other examples of anti-idiotypic antibodies include those available from AbD Commercially available ones, anti-idiotypic antibodies specific for the anti-CD22 antibody described in International Patent Publication No. WO 2013 / 188864, anti-idiotypic antibodies specific for the anti-CEA antibody described in International Patent Publication No. WO 97 / 34636, anti-idiotypic antibodies specific for the anti-GD2 antibody described in U.S. Pat. No. 5,935,821, and anti-idiotypic antibodies specific for the anti-NY-ESO-1 antibody described in Jakka et al. 2013, Anticancer Research, 33(10):4189-420. Custom anti-idiotypic antibodies can also be obtained from AbD get.

[0232] Alternatively, anti-idiotypic antibodies directed against CARs targeting CD19 or other tumor-associated antigens can be prepared according to the methods described in Jena et al. (supra) and used to construct anti-idiotypic antigen-binding polypeptide constructs.

[0233] In another embodiment, the bispecific polypeptide includes a second antigen binding protein that is bound to the extracellular region of a 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 CAR (i.e., between the transmembrane domain and the scFv portion of CAR), or to the variable heavy chain of the scFv portion of CAR and the spacer between the variable light chain, or any other region of CAR.

[0234] In some embodiments, the hinge region can be a scFv-CD28 or scFv-CD8 connection that contains a new epitope that can be targeted by a second binding protein. In some embodiments, the hinge region can contain a mutant (no Fc binding) IgG CH2 / 3 that can be targeted by a second binding protein. In some embodiments, the hinge region can contain a spacer that can be targeted by a second binding protein, such as Strep-tag II described by Liu et al. (2016, Nature Biotechnology, 34: 430-434).

[0235] An example of an anti-CAR antibody that binds to the hinge region of a CAR molecule is a 2D3 antibody that binds to the CH2-CH3 hinge region of IgG4 described in International Patent Application Publication No. WO 2014 / 190273. In some embodiments, the multispecific antigen-binding construct comprises an antigen-binding polypeptide construct that binds to the CH2-CH3 hinge region of IgG4. In some embodiments, the multispecific antigen-binding construct comprises an antigen-binding polypeptide construct that binds to the CH2-CH3 hinge region of IgG4 and has one or more of the same CDRs as 2D3 (i.e., one or more or all of VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3) or an antigen-binding polypeptide construct having one or more (e.g., two) variable regions of 2D3 as described in WO 2014 / 190273. 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 as described in WO 2014 / 190273.

[0236] In another embodiment, the second antigen-binding protein of the bispecific polypeptide binds to an antigen on an immune cell expressing CAR, wherein the antigen is not CAR or is not on CAR.For example, antigens may include naturally occurring proteins (such as CD3, CD4, CD8, CD25, CD127, CD196 (CCR6), CD27, CD28, CD45RA, CD45RO, CD62L, CD197 and HLA-DR) present on the surface of immune cells. Alternatively, the antigen on the immune cell may be an artificially introduced antigen, such as an affinity tag.Examples of peptide tags that may be present on the surface of immune cells include peptide tags (e.g., FLAG- tags, HA- tags, His- tags, Myc- tags, S- tags, SBP- tags, Strep- tags, eXact- tags) and protein tags (e.g., GST- tags, MBP- tags, GFP- tags).

[0237] The first and second antigen-binding proteins of the bispecific polypeptides used in the methods of the present invention can be in the form of antibodies or antibody fragments. In one embodiment, the first antigen-binding protein comprises scFv. In another embodiment, the second antigen-binding protein comprises scFv. In certain embodiments, the first and second antigen-binding proteins are in the form of different antibodies or antibody fragments. Alternatively, the first and second antigen-binding proteins can be in the form of the same antibody or antibody fragment. In certain instances, the first antigen-binding protein (i.e., for binding to antigens on APC, preferably full-time APC) is in the form of immunoglobulins (such as IgG1, IgG2, IgG3 or IgG4), and the second antigen-binding protein is in the form of antibody fragments (such as scFv). In other instances, the second antigen-binding protein (i.e., for binding to antigens on immune cells, preferably CAR T cells) is in the form of immunoglobulins (such as IgG1, IgG2, IgG3 or IgG4), and the first antigen-binding protein (for binding to antigens on APC) is in the form of antibody fragments (such as scFv).

[0238] In a preferred embodiment, the bispecific polypeptide used according to the present invention is in the form of a fusion protein. The fusion protein preferably comprises a first antigen-binding protein connected directly or via a linker to a second antigen-binding protein. The linker can be any linker known to the skilled person for use in fusion proteins.

[0239] The linker may, for example, function to connect two proteins of an antigen-binding polypeptide construct (such as VH and VL of a scFv or a double antibody), or they may function to connect two antigen-binding polypeptide constructs (such as two or more Fabs or sdAbs) together, or they may function to connect an antigen-binding polypeptide construct to a scaffold. In some embodiments, the bispecific polypeptide may comprise a plurality of (i.e., two or more) linkers, for example, one or more scFvs connected to a scaffold may comprise a linker connecting the VH and VL of the scFv and a linker connecting the scFv to the scaffold. Suitable linkers are known in the art and can be easily selected by those skilled in the art 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).

[0240] Useful linkers include glycine-serine (GlySer) linkers known in the art, and include glycine and serine units combined in various orders. Examples include, but are not limited to, (GS), (GSGGS)n, (GGGS)n, and (GGGGS)n, wherein n is an integer of at least one, typically an integer between 1 and about 10, such as between 1 and about 8, between 1 and about 6, or between 1 and about 5.

[0241] Other useful linkers include sequences derived from immunoglobulin hinge sequences. The linker may include all or part of a hinge sequence from any of the four IgG categories, and may optionally include additional sequences. For example, the linker may include a portion of an immunoglobulin hinge sequence and a glycine-serine sequence. Non-limiting examples include approximately the first 15 residues of the IgG 1 hinge, followed by a linker having a length of about 10 amino acids of a GlySer linker sequence such as those described above.

[0242] The length of the joint will vary according to its application. Suitable joint length can be easily selected by the technician. For example, when a joint is used to connect the VH and VL domains of scFv, the length of the joint is generally between about 5 and about 20 amino acids, for example, the length is between about 10 to about 20 amino acids, or the length is between about 15 to about 20 amino acids. When a joint is used to connect the VH and VL domains of a double antibody, the joint should be short enough to prevent the two domains from associating in the same chain. For example, the length of the joint can be between about 2 and about 12 amino acids, such as, the length is between about 3 and about 10 amino acids, or the length is about 5 amino acids.

[0243] In some embodiments, when a linker is used to connect two Fab fragments, the linker can be selected so that it maintains the relative spatial conformation of the paratope of the F(ab') fragment and is able to form a covalent bond equivalent to the disulfide bond in the core hinge of IgG. In this context, suitable linkers include IgG hinge regions, such as, for example, those from IgG1, IgG2 or IgG4. Modified forms of these exemplary linkers can also be used. For example, modifications that improve the stability of the IgG4 hinge are known in the art (see, for example, Labrijn et al. 2009, Nature Biotechnology, 27: 767-771).

[0244] The linker can include a sequence of amino acid residues connecting the first and second antigen-binding proteins. Alternatively, the first and second binding proteins can be connected via chemical conjugation (e.g., to form a biaryl conjugate between proteins). Examples of suitable methods for chemical conjugation of binding proteins are known in the art. Such methods include the use of succinimidyl compounds to modify primary amines present on lysine residues, such as those used in TriLink Technologies bioconjugation reagents. In one embodiment, if the first and / or second binding protein is a scFv, the bispecific polypeptide comprises a linker sequence between the VH and VL proteins of the scFv. Suitable linker sequences will be known to those skilled in the art, and illustrative examples thereof include relatively flexible and hydrophilic amino acid residues.

[0245] It will be within the capabilities of the skilled artisan to determine the appropriate structure of a bispecific polypeptide for use in accordance with the present invention.

[0246] In any embodiment, the first and / or second binding domain can be in the form of an antibody or its antigen binding fragment.Antigen binding proteins can be antibodies, such as monoclonal antibodies.Antigen binding proteins can be in the form of recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR transplanted antibodies, primatized antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies, trispecific antibodies or multispecific antibodies).Antibodies can also include chemical modifications, such as conjugated to active agents or radioactive labels or reagents for improving solubility, or other modifications described herein.As used herein, antigen binding proteins can be variable domains.

[0247] The first and second antigen-binding proteins of the bispecific polypeptide are preferably in the form of an antibody or its antigen-binding fragment. In any embodiment, the first antigen-binding protein and / or the second antigen-binding protein are in the form of an antibody such as immunoglobulin G (IgG1, IgG2, IgG3 or IgG4). Optionally, the first antigen-binding protein is in the form of an antibody such as immunoglobulin G (IgG1, IgG2, IgG3 or IgG4), and the second antigen-binding protein is in the form of an antibody fragment (such as scFv, dimeric scFv (di-scFv), Fab, Fv, F (ab') 2), wherein the second antigen-binding protein is connected to the first antigen-binding protein via the C-terminus or N-terminus of the first antigen-binding protein or via an internal region, preferably wherein the second antigen-binding protein is connected to the first antigen-binding protein 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 such as immunoglobulin G (IgG1, IgG2, IgG3 or IgG4), and the first antigen binding protein is in the form of an antibody fragment (such as scFv, dimeric scFv (di-scFv), Fab, Fv, F(ab')2), wherein the first antigen binding protein is linked to the second antigen binding protein via the C-terminus or N-terminus or an internal region of the second antigen binding protein, preferably wherein 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.

[0248] In any embodiment, the bispecific polypeptide used 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 epitopes or affinity tags include peptide tags (e.g., FLAG-tags, HA-tags, His-tags, Myc-tags, S-tags, SBP-tags, Strep-tags, eXact-tags) and protein tags (e.g., GST-tags, MBP-tags, GFP-tags). In one embodiment, the epitope or affinity tag is a His tag.

[0249] In any embodiment, the bispecific polypeptide comprises a first antigen binding protein for binding to CD40. In any embodiment, the bispecific polypeptide comprises a second antigen binding protein for binding to a FLAG tag.

[0250] In any embodiment, the bispecific polypeptide comprises a first antigen binding protein and a second antigen binding protein, wherein the first antigen binding protein specifically binds CD40, and wherein the second antigen binding protein specifically binds a FLAG tag.

[0251] Preferably, the first antigen binding protein binds to the outer A-module of the membrane-proximal domain (D1a) of CD40, optionally wherein said binding does not prevent, reduce or inhibit CD40-CD40L binding.

[0252] In one embodiment the first antigen binding protein comprises an antigen binding domain comprising:

[0253] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4, and

[0254] FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0255] in:

[0256] FR1, FR2, FR3 and FR4 are each a framework region;

[0257] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0258] FR1a, FR2a, FR3a and FR4a are each framework regions;

[0259] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0260] The sequence of any one of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1 for CD40 binding proteins.

[0261] In any embodiment, CDR1, CDR2 and CDR3 refer to the complementary determining regions of the variable heavy chain (VH) of an antibody, and CDR1a, CDR2a and CDR3a are complementary determining regions of the variable light chain (VL) of an antibody, or in the case where CDR1, CDR2 and CDR3 are complementary determining regions from VL, CDR1a, CDR2a and CDR3a are complementary determining regions from VH. In such examples, the CDRs may be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, as appropriate.

[0262] Reference herein to a protein or antibody that "binds" to CD40 provides literal support for a protein or antibody that "specifically binds" to CD40.

[0263] In any embodiment, the first antigen binding protein competitively inhibits binding of an antibody or antigen binding fragment thereof to CD40, wherein the antibody or antigen binding fragment thereof comprises a VH and a VL, wherein the VH comprises the sequence set forth in SEQ ID NO: 1 and the VL comprises the sequence set forth in SEQ ID NO: 2.

[0264] In any embodiment the first antigen binding protein comprises a CDRH1 , CDRH2 and / or CDRH3 having an antigen binding domain with a variable heavy domain as defined in SEQ ID NO:1.

[0265] In any embodiment the first antigen binding protein comprises CDRL1, CDRL2 and / or CDRL3 having an antigen binding domain of a variable light chain as defined in SEQ ID NO:2.

[0266] In any embodiment the first antigen binding protein comprises CDR1 , CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined by SEQ ID NO: 1 and a variable light chain as defined by SEQ ID NO:2.

[0267] In any embodiment the first antigen binding protein comprises:

[0268] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0269] The sequence of any one of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1 below.

[0270] As defined herein, a linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0271] In any embodiment the first antigen binding protein comprises:

[0272] (i) VH, wherein the VH comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 3, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 4, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 5, and

[0273] (ii) VL, wherein the VL comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 6, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 7, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 8;

[0274] or

[0275] (iii) a VH comprising a complementarity determining region (CDR) 1, a CDR2, and a CDR3, wherein the CDR1 comprises 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 of SEQ ID NO:3, or consists thereof, and the CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence of SEQ ID NO:4, or consists thereof; and the CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence of SEQ ID NO:5. NO:5 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 or consisting of the sequence shown in NO:5, and

[0276] (iv) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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 of SEQ ID NO:6, and the CDR2 comprises 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 of SEQ ID NO:6 NO:7 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 shown in NO:7, or consists of a sequence thereof, and the CDR3 comprises 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 shown in SEQ ID NO:8 or consists of a sequence thereof.

[0277] In any embodiment, the first antigen binding protein comprises:

[0278] (i) VH, the VH comprising a complementarity determining region (CDR) 1, CDR2 and CDR3, the CDR1 comprising or consisting of the sequence shown in SEQ ID NO: 9, the CDR2 comprising or consisting of the sequence shown in SEQ ID NO: 10, the CDR3 comprising or consisting of the sequence shown in SEQ ID NO: 11, and

[0279] (ii) VL, wherein the VL comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 12, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 13, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 8;

[0280] or

[0281] (iii) a VH comprising a complementarity determining region (CDR) 1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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, and the CDR2 comprises 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 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 shown in SEQ ID NO:10, or consisting of a sequence thereof, wherein the CDR3 comprises 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 shown in SEQ ID NO:11, or consisting of a sequence thereof, and

[0282] (iv) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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 of SEQ ID NO: 12, and the CDR2 comprises 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 of SEQ ID NO: 12 NO:13 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 shown in NO:13, or consists of a sequence thereof, and the CDR3 comprises 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 shown in SEQ ID NO:8, or consists of a sequence thereof.

[0283] In any embodiment, FR1, FR2, FR3 and FR4 may refer to the framework region from the variable heavy chain (VH) of an antibody, FR1a, FR2a, FR3a and FR4a may refer to the framework region from the variable light chain (VL) of an antibody, or wherein FR1, FR2, FR3 and FR4 are framework regions from VL, and FR1a, FR2a, FR3a and FR4a are framework regions from VH. In such examples, FR may be referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3 and FR L4, as appropriate.

[0284] In one embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR H1, FR H2, FR H3 and / or FR H4 (according to Kabat or IMGT numbering) having an antigen binding domain of a variable heavy chain as defined in SEQ ID NO: 1.

[0285] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR L1, FR L2, FR L3 and / or FR L4 (according to Kabat or IMGT numbering) having an antigen binding domain of a variable light chain as defined in SEQ ID NO:2.

[0286] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR1, FR2, FR3 and / or FR4 (according to Kabat or IMGT numbering) of an antigen binding domain having a variable heavy chain as defined by SEQ ID NO:1 and FR1, FR2, FR3 and / or FR4 (according to Kabat or IMGT numbering) of an antigen binding domain having a variable light chain as defined by SEQ ID NO:2.

[0287] In any embodiment, the first antigen binding protein comprises a heavy chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1, or 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 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 shown in SEQ ID NO:1; optionally wherein the amino acid substitutions are not in a CDR and / or the antigen binding protein retains the ability to bind to CD40.

[0288] In any embodiment, the first antigen binding protein comprises a light chain variable domain, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or 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 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 shown in SEQ ID NO:2; optionally wherein the amino acid substitution is not in a CDR and / or the antigen binding protein retains the ability to bind to CD40.

[0289] In any embodiment, the first antigen binding protein comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence as shown in SEQ ID NO:1, 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, or consists of the same, and the light chain variable domain comprises the amino acid sequence as shown in SEQ ID NO:1. The amino acid sequence shown in NO:2, or 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 thereto, or consists of the amino acid sequence. Optionally, the heavy chain 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 shown in SEQ ID NO: 1 or 2, respectively; optionally wherein the amino acid substitution is not in a CDR and / or the antigen binding protein retains the ability to bind to CD40.

[0290] In any 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-terminus or C to N-terminus order).

[0291] In any embodiment, the first antigen binding protein can have, from N to C-terminus, VH then VL, or VL then VH, or any CDR 1, CDR 2, and CDR 3 defined herein as VH then any CDR 1, CDR 2, and CDR 3 defined herein as VL, or any CDR 1, CDR 2, and CDR 3 defined herein as VL then any CDR 1, CDR 2, and CDR 3 defined herein as VH.

[0292] In any embodiment, the first antigen binding protein may be in the form of:

[0293] (i) Single domain antibodies (sdAb);

[0294] (ii) single-chain Fv fragment (scFv);

[0295] (iii) dimeric scFv (di-scFv); or

[0296] (iv) One of (ii) or (iii) which is linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0297] Furthermore, as described herein, in any embodiment, the first antigen binding protein may be in the form of:

[0298] (i) Diabodies;

[0299] (ii) tri-antibody;

[0300] (iii) tetrabodies;

[0301] (iv) Fab;

[0302] (v) F(ab')2;

[0303] (vi) Fv;

[0304] (vii) bispecific antibodies or other forms of multispecific antibodies;

[0305] (viii) One of (i) to (vii), which is linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0306] 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 present 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 such as scFv.

[0307] In any embodiment, the second antigen binding protein is used to bind to the FLAG tag, and the second antigen binding protein comprises an antigen binding domain, wherein the antigen binding domain comprises:

[0308] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4, and

[0309] FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0310] in:

[0311] FR1, FR2, FR3 and FR4 are each a framework region;

[0312] CDR1, CDR2, and CDR3 are each complementarity determining regions;

[0313] FR1a, FR2a, FR3a and FR4a are each framework regions;

[0314] CDR1a, CDR2a, and CDR3a are each complementarity determining regions;

[0315] Wherein the sequence of any of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1 herein.

[0316] In any embodiment, CDR1, CDR2 and CDR3 refer to the complementary determining regions of the variable heavy chain (VH) of an antibody, and CDR1a, CDR2a and CDR3a are complementary determining regions of the variable light chain (VL) of an antibody, or in the case where CDR1, CDR2 and CDR3 are complementary determining regions from VL, CDR1a, CDR2a and CDR3a are complementary determining regions from VH. In such examples, the CDRs may be referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, as appropriate.

[0317] In any 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., 2×FLAG, 3×FLAG, etc.).

[0318] In any embodiment, the second antigen binding protein is used to specifically bind 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 wherein the additional amino acids in the FLAG tag are hydrophilic amino acids (e.g., R (Arg), D (Asp), E (Glu) and K (Lys)) and / or amino acids with aromatic side chains (e.g., Y (Tyr), F (Phe), H (His) and W (Trp)).

[0319] In a preferred embodiment, the second antigen binding protein is capable of specifically binding to a FLAG-tag that contains, comprises or consists of the following 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).

[0320] As used herein, the term FLAG-tag also encompasses FLAG-tags modified by amino acid insertions, deletions or substitutions, such as those derived from the above-mentioned FLAG-tags, in particular tags having the sequence DYKDDDDK.

[0321] Reference herein to a protein or antibody that "binds" a FLAG tag provides literal support for a protein or antibody that "specifically binds" a FLAG tag.

[0322] In any embodiment, the FLAG tag is present at the N-terminus, C-terminus, or within a protein to which the bispecific polypeptide of the invention is capable of binding, binds, or specifically binds.

[0323] In any embodiment, the second antigen binding protein competitively inhibits binding of an antibody to a FLAG tag, wherein the antibody comprises 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.

[0324] In any embodiment, the second antigen binding protein comprises a CDRH1, CDRH2 and / or CDRH3 having an antigen binding domain of a variable heavy chain as defined in SEQ ID NO:15.

[0325] In any embodiment, the second antigen binding protein comprises CDRL1, CDRL2 and / or CDRL3 having an antigen binding domain of a variable light chain as defined in SEQ ID NO:16.

[0326] In any embodiment, the second antigen binding protein comprises CDR1, CDR2 and / or CDR3 of an antigen binding domain having a variable heavy chain as defined by SEQ ID NO:15 and a variable light chain as defined by SEQ ID NO:16.

[0327] In any embodiment the second antigen binding protein comprises:

[0328] FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a,

[0329] The sequence of any one of the framework regions and / or complementarity determining regions is as described herein, preferably as described in Table 1.

[0330] As defined herein, a linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0331] In any embodiment, the second antigen binding domain comprises an antigen binding domain comprising:

[0332] (a) VH, wherein the VH comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists 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: 17; the CDR2 comprises 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: 17; NO:18 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:18; the CDR3 comprises or consists 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:19, and

[0333] (b) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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: 20; and the CDR2 comprises 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: 20. : the CDR3 comprises or consists 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:21; the CDR3 comprises or consists 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;

[0334] or

[0335] (c) a VH comprising complementary determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists 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: 23; and CDR2 comprises 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: 23. NO:24 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:24; the CDR3 comprises or consists 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:25, and

[0336] (d) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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: 26; and the CDR2 comprises 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: 26. : the CDR3 comprises or consists 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; the CDR3 comprises or consists 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;

[0337] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising:

[0338] (a) VH, wherein the VH comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17; the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18; and the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and

[0339] (b) VL, wherein the VL comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 20; the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 21; and the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 22;

[0340] or

[0341] (c) VH, wherein the VH comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 23; the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 24; the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and

[0342] (d) VL, wherein the VL comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26; the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27; and the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 22.

[0343] In any embodiment, FR1, FR2, FR3 and FR4 may refer to the framework region from the variable heavy chain (VH) of an antibody, FR1a, FR2a, FR3a and FR4a may refer to the framework region from the variable light chain (VL) of an antibody, or wherein FR1, FR2, FR3 and FR4 are framework regions from VL, and FR1a, FR2a, FR3a and FR4a are framework regions from VH. In such examples, FR may be referred to as FR H1, FR H2, FR H3, FR H4, FR L1, FR L2, FR L3 and FR L4, as appropriate.

[0344] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR H1, FR H2, FR H3 and / or FR H4 from a human germline, wherein the human germline is IGHV1-46*01 or IGHV7-4-1*02.

[0345] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR L1, FR L2, FR L3 and / or FR L4 from a human germline, wherein the human germline is IGKV2-30*01 or IGKV4-1*01.

[0346] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising: FR H1, FR H2, FR H3 and / or FR H4 from a human germline, wherein the human germline is IGHV1-46*01 or IGHV7-4-1*02; and FR L1, FR L2, FR L3 and / or FR L4 from a human germline, wherein the human germline is IGKV2-30*01 or IGKV4-1*01.

[0347] In one embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR H1, FR H2, FR H3 and / or FR H4 (according to Kabat or IMGT numbering) having an antigen binding domain of a variable heavy chain as defined in SEQ ID NO: 15.

[0348] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR L1, FR L2, FR L3 and / or FR L4 (according to Kabat or IMGT numbering) having an antigen binding domain of a variable light chain as defined in SEQ ID NO: 16.

[0349] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising FR1, FR2, FR3 and / or FR4 (according to Kabat or IMGT numbering) of an antigen binding domain having a variable heavy chain as defined by SEQ ID NO: 15 and FR1, FR2, FR3 and / or FR4 (according to Kabat or IMGT numbering) of an antigen binding domain having a variable light chain as defined by SEQ ID NO: 16.

[0350] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain comprising the amino acid sequence shown 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. 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 shown in SEQ ID NO: 15; optionally wherein the amino acid substitution is not in a CDR and / or the antigen-binding protein retains the ability to bind to a FLAG tag.

[0351] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising a variable light chain comprising the amino acid sequence shown 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. 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 shown in SEQ ID NO: 16; optionally wherein the amino acid substitution is not in the CDR and / or the antigen-binding protein retains the ability to bind to the FLAG tag.

[0352] In any embodiment, the second antigen binding protein comprises an antigen binding domain comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:15, and the variable light chain comprising the amino acid sequence shown 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. 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 sequence shown in SEQ ID NO: 15 or 16, respectively; optionally wherein the amino acid substitutions are not in the CDRs and / or the antigen-binding protein retains the ability to bind to a FLAG tag.

[0353] As described herein, the second antigen binding protein may be in the form of:

[0354] (i) Single domain antibodies (sdAb);

[0355] (ii) single-chain Fv fragment (scFv);

[0356] (iii) dimeric scFv (di-scFv);

[0357] (iv) one of (ii) or (iii) which is linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody;

[0358] (v) one of (i) to (iv) linked to a protein that binds to immune effector cells;

[0359] (vi) One of (i) to (iv) linked to a modified immune cell receptor such as a modified T cell receptor.

[0360] Additionally, as described herein, the second antigen binding protein may be in the form of:

[0361] (i) Diabodies;

[0362] (ii) tri-antibody;

[0363] (iii) tetrabodies;

[0364] (iv) Fab;

[0365] (v) F(ab')2;

[0366] (vi) Fv;

[0367] (vii) bispecific antibodies or other forms of multispecific antibodies;

[0368] (viii) One of (i) to (vii), which is linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0369] In a preferred embodiment, the second antigen binding protein is in the form of a scFv and the first antigen binding protein is in the form of an immunoglobulin G (IgG) antibody.

[0370] In any embodiment of any aspect of the invention, the bispecific polypeptide comprises, consists essentially of or consists of the amino acid sequences of SEQ ID NOs: 42 and 43.

[0371] 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.

[0372] Nucleic acids and vectors

[0373] In another aspect disclosed herein, a nucleic acid is provided that encodes a bispecific polypeptide for use in the methods described herein. In yet another aspect, a cell is provided that comprises a vector for use in the methods described herein. For example, it should be understood that in certain embodiments, the bispecific polypeptide is not directly administered to a subject in need of treatment, but can be expressed in vivo in a subject from a nucleic acid sequence or construct provided in the subject.

[0374] 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 generally refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides or modified forms or either type of nucleotide) of at least 10 bases in length. The term includes single-stranded and double-stranded forms of RNA and DNA.

[0375] In another aspect, a vector is provided, comprising a nucleic acid described herein operably linked to a regulatory sequence.

[0376] 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 suitable for use in eukaryotic cells include promoters, polyadenylation signals, transcription enhancers, translation enhancers, leader or trailer sequences that regulate 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.

[0377] 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 regulatory proteins or translational regulatory proteins, upstream open reading frames, ribosome 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 a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter.

[0378] The promoter sequence under consideration may be native to mammalian cells, or may be derived from other sources where the region is functional in the selected organism. The choice of promoter will vary depending on the intended host cell. For example, promoters that can be used for expression in mammalian cells include: metallothionein promoters, which can be induced in response to heavy metals such as cadmium; -actin promoters; and viral promoters such as SV 40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, Rous sarcoma virus LTR promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus promoter and HPV promoter, especially HPV upstream regulatory region (URR) and the like. All of these promoters are well described in the art and are readily available.

[0379] Enhancer elements can also be used herein to increase the expression level of nucleic acid sequences in vector constructs. Examples include: SV40 early gene enhancers, such as Dijkema et al. (1985, EMBO Journal, 4: 761) described in; enhancers / promoters derived from the long terminal repeats (LTR) of Rous sarcoma virus, such as Gorman et al. (1982, Proceedings of the National Academy of Science. USA, 79: 6777) described in; and elements derived from human CMV, such as Boshart et al. (1985, Cell, 41: 521) described in, such as elements contained in CMV intron A sequences.

[0380] The vector construct may also include a 3' non-translated sequence. The 3' non-translated sequence refers to a portion of a gene that contains a DNA segment that contains a polyadenylation signal and any other regulatory signal that can affect mRNA processing or gene expression. The polyadenylation signal is characterized by achieving the addition of a polyadenylic acid segment to the 3' end of the mRNA precursor. The polyadenylation signal is usually identified by homology to the classical form 5'AATAAA-3', but variations are not uncommon. The 3' non-translated regulatory DNA sequence preferably contains about 50 to 1,000 nucleotides and may contain transcription and translation termination sequences in addition to the polyadenylation signal and any other regulatory signal that can affect mRNA processing or gene expression.

[0381] As used herein, the term "operably linked" refers 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 a positioning and / or orientation of the regulatory sequence relative to the coding sequence to permit expression of the coding sequence under conditions compatible with the regulatory sequence.

[0382] 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 used in accordance with the method of the present invention and the technology of inserting nucleic acid constructs are known in the art, and illustrative examples thereof include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., slow viruses, retroviruses, adenoviruses and adeno-associated viruses).

[0383] Methods for producing bispecific polypeptides

[0384] The bispecific polypeptides used in the methods described herein can be produced using any number of expression systems known to those of skill in the art, illustrative examples of which include production in or by bacteria (e.g., E. coli, P. mirabilis), fungi (e.g., S. cerevisiae, P. pastoria, T. reesei), plants or plant cells, insects or insect cells (e.g., SF-9, SF21, Hi-5), or mammalian cells. In one embodiment, the expression system is a mammalian expression system. Suitable mammalian expression systems are known to those of skill in the art, illustrative examples of which include CHO or 293 expression systems. These expression systems are widely available from commercial suppliers. In one embodiment, the bispecific polypeptides are produced using a mammalian expression system.

[0385] Chemically conjugated bispecific polypeptides can be formed following modification of primary amines present on lysine residues with succinimidyl compounds, as described elsewhere herein.

[0386] It will be within the capabilities of the skilled artisan to utilize commercially available antibodies or antigen binding domains thereof to generate bispecific polypeptides for use in the methods as described herein.

[0387] In addition, based on the public sequence information related to the antibody or antigen-binding protein with the desired specificity, the antibody or its antigen-binding protein is replicated and such antibody or its antigen-binding protein is included in the bispecific polypeptide for the method according to the present invention. In particular, it should be understood that the method of the present invention covers the production of any bispecific polypeptide, which is designed to have the first and second antigen-binding proteins as described herein (i.e., for binding antigen-presenting cells, preferably professional antigen-presenting cells such as dendritic cells on antigens first binding protein; and for binding to antigens on immune cells expressing CAR (including wherein the antigen is an antigen on CAR) second binding protein) specific binding affinity. Technicians will understand that any combination of antigen-binding proteins with desired binding specificity can be used to obtain the bispecific polypeptide of the present invention.

[0388] In addition, the binding capacity of any bispecific polypeptide prepared for use in the method according to the prior art is assessed within the technical scope of those skilled in the art. For example, methods for assessing protein binding are known in the art, for example, as described in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). This method generally involves fixing the antigen-binding protein and contacting it with the labeled antigen. After washing to remove the non-specifically bound protein, the amount of the label is detected, and the bound antigen is therefore detected. Of course, the antigen-binding protein can be labeled and the antigen is fixed. Panning can also be used. Alternatively or additionally, surface plasmon resonance can be used for determination.

[0389] Optionally, the dissociation constant (Kd), association constant (Ka) and / or affinity constant (KD) of the immobilized antigen binding protein is determined. In one example, the "Kd" or "Ka" or "KD" of the bispecific polypeptides used in accordance with the invention is measured by a radiolabeled or fluorescently labeled ligand binding assay. In the case of "Kd", the assay equilibrates the antigen binding protein with the lowest 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.

[0390] According to another example, K d , K a or K D The expression of antigens can be measured by using a surface plasmon resonance assay, such as the BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) using immobilized antigen or immobilized antigen binding protein.

[0391] Pharmaceutical composition

[0392] In another aspect, a pharmaceutical composition for use in any method according to the present invention is provided, the pharmaceutical composition comprising the bispecific polypeptide described herein and a pharmaceutically acceptable carrier.

[0393] The compositions used herein can be prepared in a manner known in the art, and are those suitable for parenteral administration to mammals, especially humans, comprising a therapeutically effective amount of the composition and one or more pharmaceutically acceptable carriers or diluents.

[0394] As used herein, the term "pharmaceutically acceptable carrier" means any suitable carrier, diluent or excipient. These include: all aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers and solutes to make the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may contain suspending and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic agents and absorbents, etc. It should be understood that the composition used in the method of the present invention may also contain other supplementary physiologically active agents.

[0395] The carrier is generally pharmaceutically "acceptable" in the sense that it is compatible with the other ingredients in the composition and is not harmful to the subject. These compositions include those suitable for parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal administration). These compositions can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. Such methods include preparing a carrier for associating with isolated T cells. Typically, these compositions are prepared by uniformly and tightly associating any active ingredient with a liquid carrier.

[0396] In one embodiment, the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.

[0397] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bactericides and solutes to 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.

[0398] The present disclosure also contemplates the combination of compositions as described herein with other active agents and / or other treatment regimens or modes in addition thereto, such as radiotherapy or surgery. When compositions as described herein are used in combination with known active agents, the combination can be administered in sequence (continuously or disconnected during non-treatment) or simultaneously or as a mixture. Suitable anticancer agents will be known to those skilled in the art. Combination therapy also contemplates treatment with the compositions used in the methods of the present invention, followed by known treatment, or treatment with known agents, followed by treatment with the compositions used in the methods of the present invention, such as as maintenance therapy. For example, in the treatment of cancer, the compositions contemplated for use in the methods of the invention can be administered in combination with an alkylating agent (such as mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin, or a platinum-containing alkylating agent such as cisplatin, carboplatin, and oxaliplatin), an antimetabolite (such as a purine or pyrimidine analog or an antifolate such as azathioprine and mercaptopurine), an anthracycline (such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, or an anthracycline analog), a plant alkaloid (such as a vinca alkaloid or a taxane such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, or doestaxel), a topoisomerase inhibitor (such as a type I or type II topoisomerase inhibitor), a podophyllotoxin (such as etoposide or teniposide), a tyrosine kinase inhibitor (such as imatinib mesylate, nilotinib, or dapoxetine), In some embodiments, the present invention relates to an anti-PD-1 inhibitor (such as sartinib), an adenosine receptor inhibitor (such as an A2aR inhibitor, SCH58261, CPI-444, SYN115, ZM241385, FSPTP or an A2BR inhibitor such as PSB-1115), an adenosine receptor agonist (such as CCPA, IB-MECA and CI-IB-MECA), a checkpoint inhibitor (including those of the PDL-1:PD-1 axis, nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A or MSB0010718C), an inhibitor of the CTLA-4 pathway (such as ipilimumab and tremelimumab), an inhibitor of the TIM-3 pathway, or an agonist monoclonal antibody known to promote T cell function (including anti-OX40 such as MEDI6469; and anti-41BB such as PF-05082566).

[0399] In another aspect, a kit or article of manufacture for use in the methods of the present invention is provided, wherein the kit or article of manufacture comprises one or more bispecific polypeptides of the present invention, nucleic acids encoding the bispecific polypeptides and / or pharmaceutical compositions as described above.

[0400] In another aspect, a kit for use in the method of the present invention is provided, the kit comprising:

[0401] (a) a container containing the bispecific polypeptide, nucleic acid, vector or pharmaceutical composition of the invention; and

[0402] (b) have a label or package insert containing instructions for use.

[0403] The kit may also include one or more active ingredients or ingredients for treating cancer. For example, the kit may also include immune cells expressing CAR.

[0404] A "kit" or "article of manufacture" may include a container and a label or package insert about 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 effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the therapeutic composition is used to treat a selected condition. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or prophylactic composition can be used to treat cancer or other conditions described herein.

[0405] The kit may include (a) a therapeutic or preventive composition; and (b) a second container containing a second active ingredient or ingredient. The kit for use in the method of the present invention according to this embodiment may also include a package insert indicating that the composition and other active ingredients can be used to treat cancer or disorders described herein. Alternatively, or additionally, the kit may also 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. It may also contain other materials desired from a commercial and user perspective that will be known to those skilled in the art, suitable examples of which include other buffers, diluents, filters, needles, and syringes.

[0406] Example

[0407] Example 1—Multiple stimulation of CAR T cells with bispecific BEAT and MoDC or tumor

[0408] According to the methods and disclosures herein, a bispecific polypeptide comprising a first antigen binding protein for binding to CD40 and a second antigen binding protein for binding to a FLAG tag (ie, anti-CD40 / anti-FLAG BEAT) is obtained. The amino acid sequence of BEAT is provided as SEQ ID NO: 42 and 43.

[0409] CAR T cells are obtained by transducing CD3+ T cells with a nucleic acid sequence encoding a CAR containing a FLAG-tag.

[0410] CAR T cells were stimulated with high-expression mesothelin-positive tumor cells or BEAT and monocyte-derived DCs (MoDCs). Figure 1 On days 2 and 4, additional stimulators (tumor or MoDC) were added to each condition. On day 6, activation and exhaustion markers ( Figure 2 The phenotypes of all conditions were also evaluated ( Figure 3 ).

[0411] Compared to tumor-stimulated CAR T cells, the CAR T cell + BEAT and MoDC groups exhibited reduced CAR T cell exhaustion markers LAG-3, PD-1, and Tim-3, even after multiple stimulations with MoDC. In contrast, the CAR T cell + tumor group treated with multiple tumor stimulations on days 2 and 4 had increased activation (CD25, CD69) and exhaustion (LAG-3, PD-1, and Tim-3) phenotypes. In a 6-day proliferation assay using the CAR + tumor group that had not been subjected to multiple tumor stimulations, activation and exhaustion were not prevalent ( Figure 2 ).

[0412] In addition, restimulation of CAR T cells with BEAT and MoDC did not change the CAR T cell memory phenotype (TSCM and TCM) compared with CAR T cells without additional stimulation with BEAT and MoDC. However, the CAR T cell phenotype after tumor restimulation showed an increased effector memory phenotype (TEM) and reduced TSCM compared with CAR T cells without additional tumor stimulation. For CD4+ and CD8+ subsets, the TCM and TSCM CAR T cell phenotypes were also similar between CAR T cells stimulated with tumors (without restimulation) and CAR T cells stimulated with BEAT and MoDC (both with and without restimulation), although the TSCM phenotype in the CD8+ subset increased after stimulation (and restimulation) with BEAT and MoDC ( Figure 3 ).

[0413] Example 2—Bispecific BEAT rescues exhausted tumor-pretreated CAR T cells

[0414] In this experiment, CAR T cells were sorted from each stimulation condition as described in the previous examples and restimulated with BEAT and MoDC, or with mesothelin-positive or mesothelin-negative tumors. Figure 4 )

[0415] After another 5 days of culture (day 11), the phenotypes of all conditions were evaluated ( Figure 5). When CART cells were treated with mesothelin-positive tumors, BEAT- and MoDC-pretreated CAR T cells showed higher central memory (TCM) and stem cell memory (TSCM) phenotypes compared to 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 5 B). In contrast, CAR T cells that had been pretreated with mesothelin-positive tumors and restimulated with mesothelin-positive tumors ( Figure 5 Mesothelin-positive tumors restimulated with CAR T cells pre-treated with BEAT and MoDC had increased TCM and TSCM phenotypes compared with (B). Figure 5 A).

[0416] Furthermore, CAR T cells primed with tumors showed signs of exhaustion ( Figure 6 A) Compared with BEAT and MoDC pre-treated CAR T cells ( Figure 6 B) shows increased proliferation of CAR T cells. Re-stimulation with mesothelin-positive tumors or MoDC alone did not affect the 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 antigens, while no proliferation was observed in the presence of mesothelin-negative tumors ( Figure 6 ).

Claims

1. A method for rescuing immune effector cell activity or reversing immune effector cell exhaustion, the method comprising exposing immune effector cells having or suspected of having an exhausted phenotype to an effective amount of 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 antigen binding protein that specifically binds to an antigen on said immune effector cell, -optionally wherein the immune effector cell expresses a heterologous receptor comprising an antigen binding protein and a signaling protein, Thereby rescuing immune effector cell activity or reversing immune effector cell exhaustion.

2. A method for increasing immune effector cell activity or cytotoxicity of exhausted immune effector cells, the method comprising exposing a plurality of immune effector cells exhibiting one or more signs of an exhausted phenotype to an effective amount of 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 antigen binding protein that specifically binds to an antigen on said immune effector cell, -optionally wherein the immune effector cell expresses a heterologous receptor comprising an antigen binding protein and a signaling protein, Thereby increasing immune cell effector activity or cytotoxicity.

3. A method for preventing, inhibiting, reducing the progression of, or delaying the onset of immune effector cell exhaustion, the method comprising exposing a plurality of immune effector cells having or suspected of having an exhausted phenotype to an effective amount of 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 antigen binding protein that specifically binds to an antigen on said immune effector cell, -optionally wherein the immune effector cell expresses a heterologous receptor comprising an antigen binding protein and a signaling protein, Thereby preventing, inhibiting, reducing the progression of immune effector cell exhaustion or delaying the onset of immune effector cell exhaustion.

4. A method for treating a disease or condition characterized by exhaustion of immune effector cells, the method comprising administering to a subject in need thereof an effective amount of 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 antigen binding protein that specifically binds to an antigen on an immune effector cell of the subject, Diseases or conditions characterized by depletion of immune effector cells are thereby treated.

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

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

7. A method according to any one of claims 1 to 6, wherein the immune effector cell expresses a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) that binds a target antigen (such as a tumor-associated antigen).

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

9. The method of claim 1, wherein the immune effector cells are CAR T cells and the method is used to rescue CAR T cell activity or reverse CAR T cell exhaustion.

10. The method of claim 2, wherein the immune effector cell is a CAR T cell, and the method is used to increase the CAR T cell activity or cytotoxicity of an exhausted CAR T cell, preferably wherein the cell has or is suspected of having an exhausted phenotype.

11. The method of claim 3, wherein the immune effector cell is a CAR T cell, and wherein the method is used to prevent, inhibit, reduce the progression of CAR T cell exhaustion or delay the onset of CAR T cell exhaustion, preferably wherein the cell has or is suspected of having an exhausted phenotype.

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

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

14. The method of claim 13, wherein the method comprises the step of further administering to the subject the immune effector cells that have been contacted with the bispecific polypeptide.

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

16. A method comprising: - Rescue of immune effector cell (e.g. CAR T cell) activity; -Reversal of 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 -Prevent, inhibit, reduce the progression of, or delay the onset of, immune effector cell (e.g., CAR T cell) exhaustion, The method comprises administering to the subject a bispecific polypeptide 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 the immune (e.g., CAR T) cell.

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 therapy with CAR T cells or other immune effector cells.

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 more after administration of the CAR T cells (or other immune effector cell therapy) to the subject.

19. The method of any one of claims 1 to 18, wherein upon administration of the bispecific polypeptide to a subject, an exhaustion phenotype of one or more of the immune effector cells, or a marker or parameter indicative of the exhaustion phenotype, is detectable or measurable in the subject or in a biological sample from the subject.

20. The method of any one of claims 1 to 19, wherein upon administration of the bispecific polypeptide to a subject, an exhaustion phenotype of one or more of the immune effector cells, or a marker or parameter indicative of the exhaustion phenotype, has been detected or measured in the subject or in a biological sample from the subject.

21. A method according to any one of claims 1 to 19, wherein the method comprises determining the risk or likelihood that a subject has immune effector cells (such as CAR T cells) with an exhausted phenotype.

22. A method according to claim 20, wherein determining whether a subject has or is at risk of having immune effector cells (such as CAR T cells) with an exhausted phenotype includes determining the expression of one or more genes associated with immune cell exhaustion in a sample of immune effector cells obtained from the subject, and / or determining an increase in the level or extent of surface expression of markers on the immune cells compared to a reference population of immune effector cells and / or determining the percentage of immune cells that exhibit surface expression of one or more exhaustion markers.

23. Use of a bispecific polypeptide in the manufacture of a medicament for: in a subject, -Rescue or reverse exhaustion of immune effector cells (e.g., CAR T cells); - Increase immune effector cell activity or cytotoxicity of exhausted immune effector cells (e.g., CAR T cells); or -Prevent, inhibit, reduce the progression of, or delay the onset of, immune effector cell (e.g., CAR T cell) exhaustion, Preferably wherein the subject has received or is receiving treatment with engineered immune effector cells (such as CAR T cells).

24. The method according to any one of claims 1 to 23, wherein the method is used to treat a disease or condition selected from cancer, chronic infection (such as chronic bacterial or chronic viral infection), chronic inflammation or autoimmunity.

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

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

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. 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 (such as Myc, Flag, His, HA, SBP, GST, MBP, GFP, S, Strep, eXact or other tags), or any other region of the extracellular portion of the CAR.

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

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

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

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 region or a connecting region. Suitable linkers are further described herein.

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

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

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

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

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

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

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

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, and wherein the first antigen binding protein comprises a CDRH1, CDRH2 and / or CDRH3 having an antigen binding domain of a variable heavy domain as defined in SEQ ID NO: 1 and / or a CDRL1, CDRL2 and / or CDRL3 having an antigen binding domain of a variable light chain as defined in SEQ ID NO:

2.

44. The method of any one of claims 1 to 43, wherein the first antigen binding protein comprises: FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a, Optionally, wherein the linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues; and The first antigen-binding protein comprises an antigen-binding domain, and the antigen-binding domain comprises: (i) VH, wherein the VH comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 3, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 4, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 5, and (ii) VL, wherein the VL comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 6, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 7, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:8; or (iii) a VH comprising a complementarity determining region (CDR) 1, a CDR2, and a CDR3, wherein the CDR1 comprises 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 of SEQ ID NO: 3, or consists thereof, and the CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence of SEQ ID NO: 4, or consists thereof; and the CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence of SEQ ID NO:

5. NO:5 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 or consisting of the sequence shown in NO:5, and (iv) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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 of SEQ ID NO:6, and the CDR2 comprises 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 of SEQ ID NO:6 NO:7 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 shown in NO:7, or consists of a sequence thereof, and the CDR3 comprises 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 shown in SEQ ID NO:8, or consists of a sequence thereof.

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 of SEQ ID NO: 1, or 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 thereto.

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 of SEQ ID NO:2, or 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 thereto.

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

16.

48. The method of any one of claims 1 to 47, wherein the second antigen binding protein comprises: FR1-CDR1–FR2–CDR2–FR3–CDR3–FR4–Linker-FR1a-CDR1a–FR2a–CDR2a–FR3a–CDR3a–FR4a, Optionally wherein the linker can be a chemical, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues; and Wherein the second antigen binding domain comprises an antigen binding domain, wherein the antigen binding domain comprises: (i) VH, wherein the VH comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 23, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 24, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO: 25, and ii) VL, wherein the VL comprises a complementarity determining region (CDR) 1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO: 26, the CDR2 comprises or consists of the sequence shown in SEQ ID NO: 27, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:28; or (iii) a VH comprising complementary determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises or consists 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: 23; and CDR2 comprises 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:

23. NO:24 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:24; the CDR3 comprises or consists 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:25, and (iv) a VL comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises or consists 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: 26; and the CDR2 comprises 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:

26. NO:27 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, or consists 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:22, or consists 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:

22.

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

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