Immunoconjugate molecules and related methods and compositions

Abstract

The immunoconjugate molecules contain an interleukin-2 (IL-2) polypeptide and a masking moiety that can inhibit and activate IL-2 activity under suitable conditions. Methods for producing the immunoconjugate molecules. Therapeutic uses of the immunoconjugate molecules due to their modulating effect on the immune system to treat diseases such as cancer and other chronic infectious diseases. The present disclosure provides immunoconjugate molecules comprising cytokine polypeptides. The present disclosure also provides, in certain embodiments, polynucleotides and vectors comprising sequences encoding such immunoconjugate molecules, as well as compositions, reagents, and kits comprising such immunoconjugate molecules.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from PCT / CN2021 / 100705, filed June 17, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to Electronically Submitted Sequence Listings This application contains a Sequence Listing that has been submitted electronically as an ASCII formatted Sequence Listing in the file "14625-006-228_SEQLIST.txt" having a size of 90,098 bytes, with a creation date of May 10, 2022. The electronically submitted Sequence Listing is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] 1. Field The present disclosure relates generally to interleukin-2 (IL-2)-containing immunoconjugate molecules. More particularly, the present disclosure relates to immunoconjugate molecules that have demonstrated improved properties for use as immunotherapeutics due to their ability to modulate the immune system. The present disclosure further relates to therapeutic uses and pharmaceutical compositions of the immunoconjugate molecules for treating diseases such as cancer and other chronic infectious diseases. [Background technology]

[0004] 2.Background Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein that plays a central role in lymphocyte development, survival, and homeostasis. IL-2's ability to expand lymphocyte populations in vivo and increase the effector function of these cells confers antitumor effects to IL-2, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Therefore, high-dose IL-2 treatment has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. However, because IL-2 has dual functions in the immune response, not only mediating the expansion and activity of effector cells but also critically involved in maintaining peripheral immune tolerance, soluble IL-2 is not optimal for inhibiting tumor growth. An additional concern associated with IL-2 immunotherapy is the side effects caused by recombinant human IL-2 treatment. For example, patients receiving high-dose IL-2 treatment frequently experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological, and systemic adverse events, which require intensive monitoring and patient management. Thus, there remains a need in the art to further enhance the therapeutic utility of IL-2 proteins. The present disclosure satisfies this need. Summary of the Invention [Means for solving the problem]

[0005] 3. Overview The present disclosure provides immunoconjugate molecules comprising cytokine polypeptides. In certain embodiments, the present disclosure also provides polynucleotides and vectors comprising sequences encoding such immunoconjugate molecules, as well as compositions, reagents, and kits comprising such immunoconjugate molecules. In a related aspect, methods are also provided herein for using immunoconjugate molecules according to the present disclosure to deliver and / or activate cytokine activity at a target site, or to reduce toxicity and / or other side effects associated with systemic exposure to cytokine activity in a subject.

[0006] The present disclosure also provides, in certain embodiments, peptides or polypeptides, such as antibodies or antigen-binding fragments thereof, that can form part of such immunoconjugate molecules of the present disclosure. In specific embodiments, provided herein are binding proteins comprising antibodies or fragments thereof that bind to fibrosis activating proteins (FAPs). In specific embodiments, provided herein are bispecific binding proteins comprising two-in-one antibodies or fragments thereof that bind to both FAPs and interleukin-2 (IL-2).

[0007] In some embodiments, the immunoconjugate molecule of the present disclosure comprises a cytokine portion comprising a cytokine polypeptide having cytokine activity and a masking portion. Such a masking portion comprises a bispecific antibody or antigen-binding fragment thereof capable of binding to the cytokine polypeptide and a first target antigen. When the masking portion binds to the cytokine polypeptide, it reduces or inhibits cytokine activity, and when the masking portion binds to a second target antigen, it dissociates from the cytokine polypeptide, thereby activating cytokine activity.

[0008] In some embodiments, the masking moiety comprises a VHH formed from an intact antibody, a Fab, a Fab', a F(ab')2, an Fv, a scFv, a dsFv, a diabody, a triabody, a tetrabody, or an antibody fragment. In some embodiments, the bispecific antibody is a two-in-one antibody.

[0009] In some embodiments, the first target antigen is not a cytokine polypeptide. In some embodiments, the first target antigen is expressed on a cell surface. In some embodiments, the cell is a cancer cell or a cell in the tumor microenvironment. In some embodiments, the first target antigen is soluble. In some embodiments, the first target antigen is a tumor-associated antigen. In some embodiments, the first target antigen is a fibrosis activating protein (FAP).

[0010] In some embodiments, the cytokine portion comprises wild-type or mutant interleukin-2 (IL-2). In some embodiments, the cytokine portion comprises human IL-2 or mutant human IL-2.

[0011] In some embodiments, the immunoconjugate molecule further comprises an anchoring moiety comprising an antibody or antigen-binding fragment thereof that specifically binds to a second target antigen. In some embodiments, the second target antigen is expressed on the cell surface. In some embodiments, the cell is a cancer cell or a cell in a tumor microenvironment. In some embodiments, the second target antigen is soluble. In some embodiments, the second target antigen is a tumor-associated antigen.

[0012] In some embodiments, the first and second target antigens are the same. In some embodiments, the bispecific masking moiety and the anchoring moiety bind to the same epitope of the first or second target antigen. In some embodiments, the bispecific masking moiety and the anchoring moiety bind to different epitopes of the first or second target antigen. In some embodiments, the first target antigen and the second target antigen are different. In some embodiments, the second target antigen is a fibrosis activating protein (FAP).

[0013] In some embodiments, the anchoring moiety comprises an intact antibody, a Fab, a Fab', a F(ab')2, an Fv, an scFv, a dsFv, a diabody, a triabody, a tetrabody, or a VHH formed from an antibody fragment. In specific embodiments, the bispecific antibody or antigen-binding fragment of the masking moiety is a Fab, an ScFv, or a VHH. In specific embodiments, the antibody or antigen-binding fragment of the anchoring moiety is a Fab, an ScFv, or a VHH.

[0014] In some embodiments, the immunoconjugate molecule further comprises a conjugate moiety that operably connects two or more of the cytokine moiety, the masking moiety, and the anchoring moiety of the immunoconjugate molecule.

[0015] In some embodiments, the conjugate moiety comprises an immunoglobulin Fc domain or a mutant thereof. In some embodiments, the Fc domain comprises two non-identical polypeptide chains, a first subunit and a second subunit; the Fc domain comprises a first modification that promotes heterodimerization of the two non-identical polypeptide chains. In some embodiments, the first modification is a knob-into-hole modification, comprising a knob modification in the first subunit and a hole modification in the second subunit.

[0016] In some embodiments, the Fc domain comprises a second modification, and the Fc domain has reduced binding affinity to an Fc receptor compared to a native Fc domain without said second modification. In some embodiments, the Fc domain has reduced binding affinity to an Fcγ receptor compared to a native Fc domain without said second modification. In some embodiments, the Fcγ receptor is an FcγRIIIα, FcγRI, or FcγRIIα receptor.

[0017] In some embodiments, the Fc domain has a reduced binding affinity for a complement component compared to a native Fc domain without said second modification, hi some embodiments, the complement component is C1q.

[0018] In some embodiments, the Fc domain has a reduced Fc effector function compared to an Fc domain without said second modification, hi some embodiments, the reduced Fc effector function is selected from complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, down-regulation of cell surface receptors, and B-cell activation.

[0019] In some embodiments, the second modification comprises one or more mutations selected from S228P, E233P, L234V, L234A, L235A, L235E, ΔG236, D265G, N297A, N297D, P329E, P329S, P329A, P329G, A330S, or P331S, wherein the numbering is that of the EU index as in Kabat. In some embodiments, the second modification comprises one or more mutations selected from E233P, L234V, L234A, L235A, ΔG236, D265G, P327E, A328S, P329E, A330S, or P331S, wherein the numbering is that of the EU index as in Kabat.

[0020] In some embodiments, the cytokine moiety is connected to the C-terminus of one of the first and second subunits of the Fc domain, and the masking moiety is connected to the C-terminus of the other of the first and second subunits of the Fc domain. In some embodiments, the anchoring moiety is connected to the N-terminus of one of the first and second subunits of the Fc domain. In some embodiments, the anchoring moiety and the cytokine moiety are connected to the same subunit of the Fc domain. In some embodiments, the anchoring moiety and the masking moiety are connected to the same subunit of the Fc domain. In some embodiments, the masking moiety is connected to the C-terminus of one of the first and second subunits of the Fc domain; and the cytokine moiety is connected to the masking moiety. In some embodiments, the anchoring moiety and the masking moiety are connected to the N-terminus of one of the first and second subunits of the Fc domain. In some embodiments, the anchoring moiety and the masking moiety are connected to the same subunit of the Fc domain; or the anchoring moiety and the masking moiety are connected to different subunits of the Fc domain. In some embodiments, the masking moiety is connected to the N-terminus of one of the first and second subunits of the Fc domain, and the cytokine moiety is connected to the masking moiety. In some embodiments, the masking moiety is connected to the N-terminus of one of the first and second subunits of the Fc domain, and the anchoring moiety is connected to the N-terminus of the other of the first and second subunits of the Fc domain. In some embodiments, the cytokine moiety is connected to the masking moiety. In some embodiments, the cytokine moiety is connected to the anchoring moiety.

[0021] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof of the masking moiety is a Fab, ScFv, or VHH. In some embodiments, the antibody or antigen-binding fragment thereof of the anchoring moiety is a Fab, ScFv, or VHH. In some embodiments, the connection between two or more of the cytokine moiety, masking moiety, anchoring moiety, and conjugate moiety is via a peptide linker.

[0022] In some embodiments, the cytokine is an IL-2 polypeptide. In specific embodiments, the cytokine polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1, 3, 7-15, and 107-110. In some embodiments, the first target antigen and the second target antigen are fibroblast activation proteins (FAPs). In specific embodiments, the first target antigen and the second target antigen are human FAPs.

[0023] In a specific embodiment, the masking moiety comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), wherein the antibody or antigen-binding fragment comprises: (a) a VL complementarity-determining region 1 (CDR1), a VL CDR2, and a VL CDR3 of any one of antibodies D001, D002, D029, D029LV1, D029LV2, D029LV3, D029LV4, D029LV5, D003, D047, D049, or B10 shown in Table 1; and / or (b) a light chain variable region (VH) comprising a VH complementarity-determining region 1 (CDR1), a VH CDR2, and a VH CDR3 of any one of antibodies D001, D002, D029, D029HV1, D029HV2, D029HV3, D029HV4, D029HV5, D029HV6, D003, D047, D049, or B10 as shown in Table 2.

[0024] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively.

[0025] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 17, and 30, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 39, and 38, respectively.

[0026] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 40, 41, and 38, respectively.

[0027] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 31, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively.

[0028] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 17, and 33, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively.

[0029] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 34, 17, and 35, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 52, 53, and 51, respectively.

[0030] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 24, 25, and 23, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 40, 42, and 38, respectively.

[0031] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 28, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 42, and 38, respectively.

[0032] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 29, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 42, and 38, respectively.

[0033] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 24, 25, and 29, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 40, 42, and 38, respectively.

[0034] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 27, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 42, and 38, respectively.

[0035] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 27, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 44, 42, and 38, respectively.

[0036] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 27, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 45, 42, and 38, respectively.

[0037] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 103, 17, and 104, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 105, 106, and 38, respectively.

[0038] In some embodiments, the masking moiety comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), wherein the antibody or antigen-binding fragment comprises: (a) a light chain variable region (VL) comprising the VL of any one of antibodies D001, D002, D029, D029LV1, D029LV2, D029LV3, D029LV4, D029LV5, D003, D047, D049, or B10 shown in Table 3; and / or (b) a heavy chain variable region (VH) comprising the VH of any one of antibodies D001, D002, D029, D029LV1, D029LV2, D029LV3, D029LV4, D029LV5, D003, D047, D049, or B10 shown in Table 4.

[0039] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:101.

[0040] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:102.

[0041] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 68; and a VH comprising the amino acid sequence of SEQ ID NO: 79.

[0042] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 69; and a VH comprising the amino acid sequence of SEQ ID NO: 80.

[0043] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 70; and a VH comprising the amino acid sequence of SEQ ID NO: 81.

[0044] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 76; and a VH comprising the amino acid sequence of SEQ ID NO: 88.

[0045] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 77; and a VH comprising the amino acid sequence of SEQ ID NO: 89.

[0046] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 78; and a VH comprising the amino acid sequence of SEQ ID NO: 90.

[0047] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 71; and a VH comprising the amino acid sequence of SEQ ID NO: 82.

[0048] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 73; and a VH comprising the amino acid sequence of SEQ ID NO: 83.

[0049] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 74; and a VH comprising the amino acid sequence of SEQ ID NO: 83.

[0050] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 75; and a VH comprising the amino acid sequence of SEQ ID NO: 82.

[0051] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 72; and a VH comprising the amino acid sequence of SEQ ID NO: 84.

[0052] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 73; and a VH comprising the amino acid sequence of SEQ ID NO: 85.

[0053] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 72; and a VH comprising the amino acid sequence of SEQ ID NO: 87.

[0054] In some embodiments, the masking portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 101; and a VH comprising the amino acid sequence of SEQ ID NO: 102.

[0055] In some embodiments, the anchoring moiety comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), wherein the antibody or antigen-binding fragment comprises: (a) a light chain variable region (VH) comprising VL complementarity determining region 1 (CDR1), VL CDR2, and VL CDR3 of any one of antibodies 872-5, 872-59, 872-70, or 872-5V1 shown in Table 5; and / or (b) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1), VH CDR2, and VH CDR3 of any one of antibodies 872-5, 872-59, 872-70, 872-5V1, or VHH6 shown in Table 6.

[0056] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 54, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 59, and 60, respectively.

[0057] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 55, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 61, 62, and 48, respectively.

[0058] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 56, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 63, and 38, respectively.

[0059] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 57, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 64, and 51, respectively.

[0060] In some embodiments, the anchoring moiety comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises an antibody that is a VHH comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 65, 66, and 67, respectively.

[0061] In some embodiments, the anchoring moiety comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises: (a) a light chain variable region (VL) comprising the VL of any one of antibodies 872-5, 872-59, 872-70, or 872-5V1 shown in Table 7; and / or (b) a heavy chain variable region (VH) comprising the VH of any one of antibodies 872-5, 872-59, 872-70, 872-5V1, or VHH6 shown in Table 8.

[0062] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:94.

[0063] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, or SEQ ID NO:99.

[0064] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 91; and a VH comprising the amino acid sequence of SEQ ID NO: 95.

[0065] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 92; and a VH comprising the amino acid sequence of SEQ ID NO: 96.

[0066] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 93; and a VH comprising the amino acid sequence of SEQ ID NO: 97.

[0067] In some embodiments, the anchoring portion comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 94; and a VH comprising the amino acid sequence of SEQ ID NO: 98.

[0068] In some embodiments, the anchoring moiety comprises an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), and the antibody or antigen-binding fragment comprises a VHH comprising the amino acid sequence of SEQ ID NO:99.

[0069] The present disclosure, in certain embodiments, provides a composition comprising an immunoconjugate molecule according to the present disclosure and a pharmaceutically acceptable carrier.

[0070] In certain embodiments, the present disclosure provides a polynucleotide encoding an immunoconjugate molecule according to the present disclosure, or a subunit or fragment thereof. In some embodiments, the polynucleotide is operably linked to a promoter. Also provided herein is a population of polynucleotides encoding an immunoconjugate molecule according to the present disclosure, or a subunit or fragment thereof. For example, in some embodiments, a first polynucleotide encodes a first subunit or polypeptide that forms part of the immunoconjugate molecule, and a second polynucleotide encodes a second subunit or polypeptide that forms part of the immunoconjugate molecule. In some embodiments, the first polynucleotide is operably linked to a first promoter, and the second polynucleotide is operably linked to a second promoter.

[0071] In certain embodiments, the present disclosure provides a vector comprising a polynucleotide according to the present disclosure. In certain embodiments, the present disclosure further provides a population of vectors comprising: (a) a first vector comprising a nucleotide sequence operably linked to a first promoter, the first vector encoding a first subunit or polypeptide forming part of the immunoconjugate molecules provided herein, and (b) a second vector comprising a nucleotide sequence operably linked to a second promoter, the second vector encoding a second subunit or polypeptide forming part of the immunoconjugate molecules provided herein.

[0072] The present disclosure provides, in certain embodiments, a cell comprising a polynucleotide according to the present disclosure. Also provided herein is a cell comprising a vector or a population of vectors according to the present disclosure. The present disclosure provides, in certain embodiments, an isolated cell that produces an immunoconjugate molecule according to the present disclosure.

[0073] Also provided herein is a population of cells comprising: (a) a first host cell comprising a polynucleotide comprising a nucleotide sequence encoding a first subunit of a polypeptide forming part of the immunoconjugate molecules provided herein, and (b) a second host cell comprising a polynucleotide comprising a nucleotide sequence encoding a second subunit of a polypeptide forming part of the immunoconjugate molecules provided herein.

[0074] Further provided herein is a population of cells comprising: (a) a first host cell comprising a polynucleotide comprising a nucleotide sequence encoding a first subunit of a polypeptide forming part of the immunoconjugate molecule provided herein operably linked to a first promoter; and (b) a second host cell comprising a polynucleotide comprising a nucleotide sequence encoding a second subunit of a polypeptide forming part of the immunoconjugate molecule provided herein operably linked to a second promoter.

[0075] The present disclosure provides, in certain embodiments, kits comprising immunoconjugate molecules according to the present disclosure.

[0076] Also provided herein are methods for producing immunoconjugate molecules according to the present disclosure, or subunits or fragments thereof. In certain embodiments, the methods comprise culturing a cell provided herein to express the immunoconjugate molecule, or subunits or fragments thereof. In other embodiments, the methods comprise expressing a polynucleotide provided herein.

[0077] In a related aspect, provided herein is a method for activating a cytokine-mediated effect at a target site, the method comprising delivering to the target site an immunoconjugate molecule comprising a cytokine and a masking moiety; the masking moiety comprises a two-in-one antibody or antigen-binding fragment thereof that binds to the cytokine through an intramolecular interaction and inhibits the cytokine-mediated effect; the two-in-one antibody or antigen-binding fragment is capable of binding to a first target antigen at the target site; when the immunoconjugate molecule is at the target site, the two-in-one antibody binds to the first target antigen and dissociates from the cytokine; and the cytokine-mediated effect is activated at the target site.

[0078] In some embodiments, the immunoconjugate molecule further comprises an anchoring moiety; the anchoring moiety comprises an antibody or antigen-binding fragment thereof capable of binding to a second target antigen at the target site.

[0079] In some embodiments, when the immunoconjugate molecule is at the target site, the antibody or antigen-binding fragment of the anchoring moiety binds to a second target antigen; the immunoconjugate molecule is immobilized at the target site.

[0080] In some embodiments, delivering the immunoconjugate molecule to the target site comprises administering the immunoconjugate molecule to a subject, hi some embodiments, cytokine activity is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% lower at a non-target site compared to cytokine activity at the target site after administration of the immunoconjugate molecule to a subject.

[0081] In a related aspect, provided herein is a method for concentrating a cytokine at a target site, comprising delivering to the target site an immunoconjugate molecule comprising the cytokine and an anchoring moiety; the anchoring moiety comprises an antibody or antigen-binding fragment thereof capable of binding to a second target antigen at the target site; the anchoring moiety binds to the second target antigen when the immunoconjugate molecule is at the target site; and the cytokine is distributed at a higher concentration at the target site compared to non-target sites.

[0082] In some embodiments, delivering the immunoconjugate molecule to the target site comprises administering the immunoconjugate molecule to a subject, hi some embodiments, cytokine concentration is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% lower at a non-target site compared to cytokine activity at the target site following administration of the immunoconjugate molecule to a subject.

[0083] In some embodiments, the immunoconjugate molecule further comprises a masking moiety; the masking moiety comprises a two-in-one antibody or antigen-binding fragment thereof that binds to the cytokine through an intramolecular interaction and inhibits the cytokine-mediated effect; the two-in-one antibody or antigen-binding fragment is capable of binding to a first target antigen at the target site; when the immunoconjugate molecule is at the target site, the two-in-one antibody binds to the first target antigen and dissociates from the cytokine; and the cytokine-mediated effect is activated at the target site.

[0084] In some embodiments, administration of the immunoconjugate molecule to a subject reduces cytokine-associated toxicity or side effects in the subject. In some embodiments, cytokine toxicity or side effects are reduced by at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% in this method compared to administration of an equivalent amount of unconjugated cytokine to a subject. In some embodiments, the reduction in cytokine-associated toxicity or side effects is measured as an extension of the lifespan of the administered subject. In some embodiments, the reduction in cytokine-associated toxicity or side effects is measured as a reduction in weight loss in the administered subject. In some embodiments, the reduction in cytokine-associated toxicity or side effects is measured as a change in the level of immune response in the administered subject. In some embodiments, the reduction in cytokine-associated toxicity or side effects is measured as a change in inflammatory response in the administered subject.

[0085] In some embodiments of the method, the first antigen and the second antigen are the same or different antigens. In some embodiments, the target site is a tumor microenvironment. In some embodiments, the target site is a cancerous cell. In some embodiments, the first and / or second antigen is expressed on the surface of a cancer cell. In some embodiments, the first and / or second antigen is expressed by a cell in the tumor microenvironment. In some embodiments, the first and / or second antigen is a fibrosis activating protein (FAP). In some embodiments, the immunoconjugate molecule further comprises a conjugate moiety configured to operably connect two or more of the cytokine polypeptide, the masking moiety, and the anchoring moiety. In some embodiments, the conjugate moiety is an immunoglobulin Fc domain comprising two non-identical polypeptide chains, a first subunit and a second subunit; the Fc domain comprises a first modification that promotes heterodimerization of the two non-identical polypeptide chains. In some embodiments, the immunoglobulin domain comprises a second modification and the Fc domain has a reduced binding affinity to an Fc receptor compared to a native Fc domain without said second modification, hi some embodiments, the immunoconjugate molecule used in the method is an immunoconjugate molecule according to the present disclosure.

[0086] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that can form part of the immunoconjugate molecule of the present disclosure. In some embodiments, a two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), wherein the antibody or antigen-binding fragment comprises: (a) a VL complementarity-determining region 1 (CDR1), a VL CDR2, and a VL CDR3 of any one of antibodies D001, D002, D029, D029LV1, D029LV2, D029LV3, D029LV4, D029LV5, D003, D047, D049, or B10 shown in Table 1; and / or (b) a heavy chain variable region (VH) comprising a VH complementarity-determining region 1 (CDR1), a VH CDR2, and a VH CDR3 of any one of antibodies D001, D002, D029, D029HV1, D029HV2, D029HV3, D029HV4, D029HV5, D029HV6, D003, D047, D049, or B10 as shown in Table 2.

[0087] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 37, and 38, respectively.

[0088] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 19, 17, and 20, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 39, and 38, respectively.

[0089] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 40, 41, and 38, respectively.

[0090] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 31, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively.

[0091] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 17, and 33, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively.

[0092] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 34, 17, and 35, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 52, 53, and 51, respectively.

[0093] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 24, 25, and 23, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 40, 42, and 38, respectively.

[0094] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 28, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 42, and 38, respectively.

[0095] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 29, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 42, and 38, respectively.

[0096] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 24, 25, and 29, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 40, 42, and 38, respectively.

[0097] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 27, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 42, and 38, respectively.

[0098] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 27, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 44, 42, and 38, respectively.

[0099] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 25, and 27, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 45, 42, and 38, respectively.

[0100] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 103, 17, and 104, respectively, and VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 105, 106, and 38, respectively.

[0101] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises: (a) a light chain variable region (VL) comprising the VL of any one of antibodies D001, D002, D029, D029LV1, D029LV2, D029LV3, D029LV4, D029LV5, D003, D047, D049, or B10 shown in Table 3; and / or (b) a heavy chain variable region (VH) comprising the VH of any one of antibodies D001, D002, D029, D029LV1, D029LV2, D029LV3, D029LV4, D029LV5, D003, D047, D049, or B10 shown in Table 4.

[0102] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 101.

[0103] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VH comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:102.

[0104] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 68; and a VH comprising the amino acid sequence of SEQ ID NO: 79.

[0105] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 69; and a VH comprising the amino acid sequence of SEQ ID NO: 80.

[0106] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 70; and a VH comprising the amino acid sequence of SEQ ID NO: 81.

[0107] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 76; and a VH comprising the amino acid sequence of SEQ ID NO: 88.

[0108] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 77; and a VH comprising the amino acid sequence of SEQ ID NO: 89.

[0109] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 78; and a VH comprising the amino acid sequence of SEQ ID NO: 90.

[0110] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 71; and a VH comprising the amino acid sequence of SEQ ID NO: 82.

[0111] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 73; and a VH comprising the amino acid sequence of SEQ ID NO: 83.

[0112] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 74; and a VH comprising the amino acid sequence of SEQ ID NO: 83.

[0113] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 75; and a VH comprising the amino acid sequence of SEQ ID NO: 82.

[0114] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 72; and a VH comprising the amino acid sequence of SEQ ID NO: 84.

[0115] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 72; and a VH comprising the amino acid sequence of SEQ ID NO: 85.

[0116] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 72; and a VH comprising the amino acid sequence of SEQ ID NO: 87.

[0117] In some embodiments, the two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2) comprises a VL comprising the amino acid sequence of SEQ ID NO: 101; and a VH comprising the amino acid sequence of SEQ ID NO: 102.

[0118] In certain embodiments, the present disclosure provides an immunoconjugate molecule comprising a two-in-one antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) and interleukin-2 (IL-2), as disclosed herein, and an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is human IL-2. In some embodiments, the IL-2 polypeptide is a wild-type or mutant IL-2 as described herein.

[0119] The present disclosure also provides, in certain embodiments, an antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP), wherein the antibody or antigen-binding fragment comprises: (a) a light chain variable region (VH) comprising a VL complementarity determining region 1 (CDR1), a VL CDR2, and a VL CDR3 of any one of antibodies 872-5, 872-59, 872-70, or 872-5V1 shown in Table 5; and / or (b) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (CDR1), a VH CDR2, and a VH CDR3 of any one of antibodies 872-5, 872-59, 872-70, 872-5V1, or VHH6 shown in Table 6.

[0120] In some embodiments, the antibody or antigen-binding fragment that binds to fibroblast activation protein (FAP) comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 54, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 59, and 60, respectively.

[0121] In some embodiments, the antibody or antigen-binding fragment that binds to fibroblast activation protein (FAP) comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 55, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 61, 62, and 48, respectively.

[0122] In some embodiments, the antibody or antigen-binding fragment that binds to fibroblast activation protein (FAP) comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 56, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 63, and 38, respectively.

[0123] In some embodiments, the antibody or antigen-binding fragment that binds to fibroblast activation protein (FAP) comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 17, and 57, respectively, and a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 58, 64, and 51, respectively.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises an antibody that is a VHH comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 65, 66, and 67, respectively.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises: (a) a light chain variable region (VL) comprising the VL of any one of antibodies 872-5, 872-59, 872-70, or 872-5V1 shown in Table 7; and / or (b) a heavy chain variable region (VH) comprising the VH of any one of antibodies 872-5, 872-59, 872-70, 872-5V1, or VHH6 shown in Table 8.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VL comprising the amino acid sequence of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:94.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VH comprising the amino acid sequence of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, or SEQ ID NO:99.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VL comprising the amino acid sequence of SEQ ID NO:91; and a VH comprising the amino acid sequence of SEQ ID NO:95.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VL comprising the amino acid sequence of SEQ ID NO:92; and a VH comprising the amino acid sequence of SEQ ID NO:96.

[0130] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VL comprising the amino acid sequence of SEQ ID NO:93; and a VH comprising the amino acid sequence of SEQ ID NO:97.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VL comprising the amino acid sequence of SEQ ID NO:94; and a VH comprising the amino acid sequence of SEQ ID NO:98.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof that binds to fibroblast activation protein (FAP) comprises a VHH comprising the amino acid sequence of SEQ ID NO:99.

[0133] In certain embodiments, the present disclosure provides an immunoconjugate molecule comprising an antibody or antigen-binding fragment thereof that binds to a fibroblast activation protein (FAP) disclosed herein and an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is human IL-2. In some embodiments, the IL-2 polypeptide is a wild-type or mutant IL-2 described herein.

[0134] In another aspect, provided herein is an immunoconjugate molecule comprising an IL-2 polypeptide conjugated to a masking moiety, wherein the masking moiety comprises a two-in-one antibody or antigen-binding fragment thereof capable of binding to the IL-2 polypeptide and a first target antigen; the masking moiety, upon binding to the IL-2 polypeptide, blocks binding of the IL-2 polypeptide to a first IL-2 receptor (IL-2R) subunit; and the masking moiety, upon binding to the first target antigen, dissociates from the IL-2 polypeptide, thereby releasing the IL-2 polypeptide for binding to the first IL-2R subunit. In some embodiments, the IL-2 polypeptide comprises one or more mutations that weaken binding of the IL-2 polypeptide to the second IL-2R subunit.

[0135] In some embodiments, the first IL-2R subunit is the IL-2R α chain (IL-2Rα) and the second IL-2R subunit is the IL-2R β chain (IL-2R β). In some embodiments, binding of the IL-2 polypeptide to the second IL-2R subunit is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to wild-type IL-2.

[0136] In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rβ are selected from D20T, D20G, D20A, H16E, H16R, H16A, N88D, N88S, N88R, V91G, V91A, V91R, and V91S, or a combination thereof. In some embodiments, the masking moiety binds to an epitope of IL-2 that includes one or more of residues P34, K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, N71, L72, Q74, Y107, and D109 of IL-2.

[0137] In some embodiments, the masking moiety binds to an epitope of IL-2 that is recognized by an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 101 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102. In some embodiments, the masking moiety competes for binding to IL-2 with an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 101 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102.

[0138] In some embodiments, the masking portion comprises (a) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDR1), VL CDR2, and VL CDR3 of antibody B10 shown in Table 1; and / or (b) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1), VH CDR2, and VH CDR3 of antibody B10 shown in Table 2.

[0139] In some embodiments, the masking portion comprises (a) a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 103, 17, and 104, respectively, and (b) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 105, 106, and 38, respectively.

[0140] In some embodiments, the masking moiety comprises (a) a light chain variable region (VL) comprising the VL of antibody B10 shown in Table 3; and / or (b) a heavy chain variable region (VH) comprising the VH of antibody B10 shown in Table 4.

[0141] In some embodiments, the masking moiety comprises a VL comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, the masking moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, the masking moiety comprises (a) a VL comprising the amino acid sequence of SEQ ID NO: 101; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 102.

[0142] In some embodiments, the first IL-2R subunit is IL-2Rβ and the second IL-2R subunit is IL-2Rα. In some embodiments, binding of the IL-2 polypeptide to IL-2Rα is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to wild-type IL-2.

[0143] In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rα are selected from K35E, R38A, R38E, R38D, F42A, F42K, K43E, Y45A, E61R, E62A, L72G, or a combination thereof. In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rα are (a) F42A; or (b) K35E and F42A. In some embodiments, the masking moiety binds to an epitope of IL-2 that includes one or more of residues L12, Q13, E15, H16, L19, D20, M23, R81, D84, D87, N88, V91, 192, and E95 of IL-2.

[0144] In some embodiments, the masking moiety binds to an epitope of IL-2 that is recognized by the antibody 5UTZ. In some embodiments, the masking moiety competes with the antibody 5UTZ for binding to IL-2.

[0145] In some embodiments, the IL-2 polypeptide further comprises one or more mutations that alter binding of the IL-2 polypeptide to the IL-2R gamma chain (IL-2Rγ). In some embodiments, the one or more mutations that alter binding of the IL-2 polypeptide to IL-2Rγ are selected from L18R, Q22E, T123A, Q126T, I129V, S130A, S130R, or a combination thereof.

[0146] In some embodiments, the immunoconjugate further comprises an anchoring moiety, wherein the anchoring moiety comprises an antibody or antigen-binding fragment thereof that specifically binds to a second target antigen. In some embodiments, the masking moiety dissociates from the IL-2 polypeptide in the presence of a first target antigen expressed on the surface of a first cell.

[0147] In some embodiments, the second target antigen is expressed on the surface of the first cell or a second cell adjacent to the first cell. In some embodiments, the first target antigen and the second target antigen are the same or different. In some embodiments, the first target antigen and / or the second target antigen is a tumor-associated antigen. In some embodiments, the first target antigen and the second target antigen are each independently selected from FAP, Her2, Her3, CD19, CD20, BCMA, PSMA, CEA, cMET, EGFR, CA-125, MUC-1, EpCAM, or Trop-2. In some embodiments, the first target antigen is a FAP.

[0148] In another aspect, provided herein is a method for activating IL-2R, comprising contacting IL-2R with an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide provided herein. In some embodiments, the IL-2R comprises IL-2Rβ. In some embodiments, the IL-2R comprises IL-2Rα. In some embodiments, the IL-2R comprises IL-2Rγ.

[0149] In some embodiments, the IL-2R comprises IL-2Rβ, and the IL-2Rβ is expressed on the surface of the first cell. In some embodiments, the IL-2R further comprises IL-2Rγ, and the IL-2Rγ is expressed on the surface of the first cell.

[0150] In some embodiments, the IL-2R further comprises IL-2Rα. In some embodiments, the IL-2Rα is associated at the cell surface. In some embodiments, the IL-2Rα is associated at the surface of a first cell (cis-presentation). In some embodiments, the IL-2Rα is associated at the surface of a second cell (trans-presentation). In some embodiments, the IL-2Rα is not associated at the cell surface. In some embodiments, the IL-2R does not comprise IL-2Rα.

[0151] In some embodiments, the first cell and / or the second cell are immune cells, and activation of IL-2R activates the immune cells. In some embodiments, immune cell activation is measured as an increase in immune cell proliferation or maturation. In some embodiments, target cell proliferation or maturation is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%. In some embodiments, immune cell activation is measured as an increase in immune cell survival time. In some embodiments, the survival time of target cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%.

[0152] In some embodiments, the immune cells are effector T cells, memory T cells, or a combination thereof. In some embodiments, the immune cells are CD4+ T cells, CD8+ T cells, helper T cells, cytotoxic T cells, SLECs (short-lived effector cells), MPECs (memory precursor effector cells), TEs (terminal effector cells), NKs (natural killer cells), NKTs (natural killer T cells), innate lymphocytes (types I-III), or a combination thereof.

[0153] In some embodiments, the immune cells are regulatory T cells (Tregs). In some embodiments, the immune cells are natural Treg (nTreg) cells, induced Treg (iTreg) cells, or a combination thereof.

[0154] In some embodiments, the first cell and / or the second cell are diseased cells, and activation of IL-2R causes the diseased cells to die. In some embodiments, the diseased cells are cancer cells. In some embodiments, the diseased cells are cells infected with an infectious pathogen. In some embodiments, the infectious pathogen is a virus, bacterium, fungus, parasite, or a combination thereof.

[0155] In one aspect, provided herein is a method of activating a target cell expressing IL-2R, the method comprising contacting the target cell with an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein, wherein upon binding of the IL-2 polypeptide to the IL-2R, the target cell is activated. In some embodiments, the target cell is an immune cell. In some embodiments, the target cell is an effector T cell, a memory T cell, a regulatory T cell, or a combination thereof. In some embodiments, the target cell is a CD4+ T cell, a CD8+ T cell, a helper T cell, a cytotoxic T cell, a SLEC (short-lived effector cell), a MPEC (memory precursor effector cell), a TE (terminal effector cell), a NK (natural killer cell), a NKT (natural killer T cell), an innate lymphocyte (type I-III), or a combination thereof. In some embodiments, the target cell is a natural Treg (nTreg) cell, an induced Treg (iTreg) cell, or a combination thereof.

[0156] In some embodiments, target cell activation is measured as an increase in target cell proliferation or maturation, hi some embodiments, target cell proliferation or maturation is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

[0157] In some embodiments, target cell activation is measured as an increase in target cell survival time, ie, an increase of about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

[0158] In some embodiments, the contacting further comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the contacting enhances an anti-neoplastic immune response. In some embodiments, the contacting enhances an anti-infectious immune response.

[0159] In one aspect, provided herein is a method for enhancing an antigen-specific immune response of a population of T cells, comprising contacting the population of T cells with an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the contacting enhances the proliferation or maturation of antigen-specific effector T cells. In some embodiments, the contacting enhances the formation of antigen-specific memory T cells. In some embodiments, the contacting is performed in the presence of an antigen. In some embodiments, the antigen is an antigen of a cancer, tumor, pathogen, or allergen.

[0160]

[0010] In one aspect, provided herein is a method of increasing secretion of a pro-inflammatory cytokine by a population of T cells, comprising contacting the population of T cells with an immunoconjugate molecule comprising an IL-2 polypeptide described herein, wherein the IL-2 polypeptide, upon binding, activates the T cells. In some embodiments, the cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof. In some embodiments, cytokine production is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%.

[0161] In one aspect, provided herein is a method for increasing the assembly of IL-2R on the surface of a target cell, the method comprising contacting the target cell with an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the IL-2R comprises IL-2Rα, IL-2Rβ, IL-2Rγ, or a combination thereof, on the surface of the target cell. In some embodiments, the IL-2R comprises IL-2Rβ and IL-2Rγ on the surface of the target cell and IL-2Rα on the surface of a second cell proximal to the target cell. In some embodiments, the IL-2R comprises IL-2Rβ and IL-2Rγ on the surface of the target cell and IL-2Rα that is not associated with the cell surface. In some embodiments, the assembly of IL-2R on the surface of target cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%. In some embodiments, the target cells are immune cells. In some embodiments, the target cells are effector T cells, memory T cells, regulatory T cells, or combinations thereof. In some embodiments, the target cells are CD4+ T cells, CD8+ T cells, helper T cells, cytotoxic T cells, SLECs (short-lived effector cells), MPECs (memory precursor effector cells), TEs (terminal effector cells), NKs (natural killer cells), NKTs (natural killer T cells), innate lymphocytes (types I-III), or combinations thereof. In some embodiments, the target cells are natural Treg (nTreg) cells, induced Treg (iTreg) cells, or combinations thereof.

[0162] In one aspect, provided herein is a method of creating a pro-inflammatory environment in a tissue surrounding a population of abnormal cells, the method comprising contacting the tissue with an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the concentration of activated B cells, CD4+ effector T cells, CD8+ effector T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and / or neutrophils in the tissue is increased. In some embodiments, the concentration of regulatory T cells in the tissue is reduced. In some embodiments, the concentration of a pro-inflammatory cytokine is increased in the tissue. In some embodiments, the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof. In some embodiments, the concentration of an antibody that binds to an antigen originating from or derived from the abnormal cells is increased in the tissue. In some embodiments, presentation of antigens originating from or derived from abnormal cells by antigen-presenting cells is increased in the tissue. In some embodiments, phagocytosis of abnormal cells is increased in the tissue. In some embodiments, apoptosis of abnormal cells induced by cell-mediated cytotoxicity is increased in the tissue. In some embodiments, apoptosis of abnormal cells induced by antibody-dependent cellular cytotoxicity is increased in the tissue. In some embodiments, the population of abnormal cells is reduced in the tissue. In some embodiments, the population of abnormal cells is reduced in the tissue by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0163] In one aspect, provided herein is a method of eliminating abnormal cells in a subject, comprising administering to the subject an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the abnormal cells are cancer cells. In some embodiments, the abnormal cells are cells infected with an infectious pathogen. In some embodiments, the infectious pathogen is a virus, bacterium, fungus, parasite, or a combination thereof.

[0164] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the treatment enhances an innate immune response, a humoral immune response, or a cell-mediated anti-neoplastic immune response. In some embodiments, the method further comprises co-administration of a second therapy.

[0165] In one aspect, provided herein is a method of treating an infectious disease in a subject in need thereof, comprising administering to the subject an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the treatment enhances an innate immune response, a humoral immune response, or a cell-mediated anti-infectious immune response. In some embodiments, the subject is co-administered with a vaccine composition to prevent the infectious disease in the subject. In some embodiments, the vaccine compositions are co-administered simultaneously or sequentially.

[0166] In one aspect, provided herein is a method of increasing a response to an antigen in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the antigen is an antigen of a cancer, tumor, pathogen, or allergen. In some embodiments, the antigen originates from or is derived from an infectious pathogen. In some embodiments, the infectious pathogen is a virus, bacterium, fungus, parasite, or a combination thereof. In some embodiments, the antigen originates from or is derived from an abnormal cell. In some embodiments, the antigen originates from or is derived from a cell infected with an infectious pathogen. In some embodiments, the infectious pathogen is a virus, bacterium, fungus, parasite, or a combination thereof. In some embodiments, the antigen originates from or is derived from a cancer cell.

[0167] In one aspect, provided herein is a method for increasing a response to a vaccine in a subject in need thereof, comprising administering to the subject an effective amount of an immunoconjugate molecule comprising a vaccine and an IL-2 polypeptide described herein. In some embodiments, the vaccine is a vaccine against a tumor, cancer, pathogen, or allergen. In some embodiments, the immunoconjugate molecule is formulated as an adjuvant composition for the vaccine.

[0168] In one aspect, provided herein is a method for establishing immune tolerance to an antigen in a tissue surrounding the antigen, the method comprising contacting the tissue with an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the concentration of activated B cells, CD4+ effector T cells, CD8+ effector T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and / or neutrophils in the tissue is reduced. In some embodiments, the concentration of regulatory T cells in the tissue is increased. In some embodiments, the concentration of a pro-inflammatory cytokine is reduced in the tissue. In some embodiments, the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof. In some embodiments, the concentration of an antibody that binds to the antigen is reduced in the tissue. In some embodiments, presentation of the antigen by antigen-presenting cells is reduced in the tissue. In some embodiments, phagocytosis of cells expressing the antigen is reduced in the tissue. In some embodiments, apoptosis of cells expressing the antigen is reduced in the tissue. In some embodiments, the tissue is in a subject and the antigen is an autoantigen to the subject. In some embodiments, the subject is suffering from an autoimmune disease.

[0169] In yet another aspect, provided herein is a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of an immunoconjugate molecule comprising an IL-2 polypeptide described herein. In some embodiments, the treatment reduces the innate immune response, humoral immune response, or cell-mediated immune response to the autoantigen. In some embodiments, the method further comprises co-administration of a second therapy. [Brief explanation of the drawings]

[0170] 4. Brief description of the drawings [Figure 1]1 is a schematic diagram of an antibody-cytokine immunoconjugate molecule according to one embodiment of the present disclosure. In an exemplary embodiment, the immunoconjugate comprises: (i) a cytokine polypeptide capable of mediating a cellular effect; (ii) a masking moiety that (a) binds to a cytokine and inhibits the cellular effect of the cytokine; (b) an antigen (e.g., a TAA) in an environment and, upon such binding, releases the cytokine; (iii) an anchoring moiety that binds to an antigen, thereby immobilizing the immunoconjugate in an antigen-enriched environment; and (iv) a conjugation moiety that connects the moieties described in (i), (ii), and (iii) of the immunoconjugate.

[0171] [Figure 2] 2 is a schematic diagram of an IL-2-containing immunoconjugate molecule according to one embodiment of the present disclosure, as well as the operation of this immunoconjugate in the absence or presence of a fibroblast activation protein (FAP). In this exemplary embodiment, the immunoconjugate comprises (i) an anti-IL-2 / anti-FAP two-in-one Fab antibody fused to the N-terminus of an immunoglobulin Fc domain, (ii) an IL-2 polypeptide fused to the N-terminus of the two-in-one antibody, and (iii) an anti-FAP antibody or binding fragment thereof fused to the N-terminus of the immunoglobulin Fc domain. The top panel shows that in the absence of a FAP in the nearby environment, the equilibrium of the two-in-one antibody shifts toward binding to IL-2 due to the prevalence of intramolecular interactions, thereby preventing IL-2 from binding to cell surface receptors and inhibiting the cellular effects of IL-2. The bottom panel shows that when immobilized in a FAP-enriched environment via binding of the anti-FAP antibody to the FAP, the equilibrium of the two-in-one antibody shifts toward dissociation from IL-2 and binding to the FAP, thereby releasing the tethered IL-2 to bind to cell surface receptors and induce cellular effects.

[0172] [Figure 3]Figure 3A shows the binding kinetics of an anti-FAP antibody designated 872-5 to biotinylated FAP immobilized on a streptavidin sensor and measured by biolayer interferometry. The K value was 6.6 nM for 872-5.

[0173] Figure 3B shows the binding kinetics of an anti-FAP antibody designated 872-59 to biotinylated FAP immobilized on a streptavidin sensor and measured by biolayer interferometry. D The value was 15.5 nM for 872-59.

[0174] Figure 3C shows the binding kinetics of an anti-FAP antibody designated 872-70 to biotinylated FAP immobilized on a streptavidin sensor and measured by biolayer interferometry. D Values ​​were <1 nM for 872-70.

[0175] [Figure 4] FIG. 4A shows the binding kinetics of a monovalent Fab-Fc fusion of D002 to biotinylated IL-2 immobilized on a streptavidin sensor and measured by biolayer interferometry.

[0176] Figure 4B shows the K D The value was 3.4 μM for the interaction of D002 with IL-2 as determined by equilibrium binding analysis.

[0177] Figure 4C shows the binding kinetics of a monovalent Fab-Fc fusion of D002 to FAP immobilized on a streptavidin sensor and measured by biolayer interferometry. D The value was 50 nM for the interaction of D002 with FAP (data not shown).

[0178] [Figure 5-1] FIG. 5A is a schematic representation of a soluble cytokine polypeptide.

[0179] 5B-5U are schematic diagrams of antibody-cytokine immunoconjugates of different molecular configurations according to the present disclosure. Specifically, FIG. 5B shows an immunoconjugate containing a cytokine polypeptide fused to the C-terminus of one of the two heavy chain fragments (e.g., the Fc-knob) of an immunoglobulin Fc domain.

[0180] FIG. 5C shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the C-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole), and (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., Fc-knob).

[0181] Figure 5D shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the C-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole) at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide fused to the C-terminus of the other of the two heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-knob), and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob).

[0182] FIG. 5E shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the C-terminus of one of the two heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole), and (b) a cytokine polypeptide fused at the N-terminus of the light chain fragment of the Fab antibody.

[0183] Figure 5F shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the C-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody; (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of an immunoglobulin Fc domain (e.g., the Fc-hole), and (c) an anti-TAA single domain antibody fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-knob).

[0184] Figure 5G shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one scFv antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one scFv antibody, (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of an immunoglobulin Fc domain (e.g., Fc-knob), and (c) an anti-TAA Fab antibody fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob).

[0185] Figure 5H shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the C-terminus of one of the two heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob), (b) a cytokine polypeptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole).

[0186] Figure 5I shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody at the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole), (b) a cytokine peptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., Fc-knob), and (c) an anti-TAA Fab fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob).

[0187] Figure 5J shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the C-terminus of one of the two heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole), (b) a cytokine peptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob).

[0188] Figure 5K shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA Fab antibody fused to the N-terminus of the other heavy chain fragment of an immunoglobulin Fc domain (e.g., the Fc-hole) at the C-terminus of the heavy chain fragment of the anti-TAA Fab antibody.

[0189] Figure 5L shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole), (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., Fc-knob), and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole).

[0190] Figure 5M shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the C-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-hole) at the N-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) a cytokine peptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (the Fc-knob), and (c) an anti-TAA scFv fused to the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody.

[0191] Figure 5N shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA Fab antibody fused to the N-terminus of the other heavy chain fragment of an immunoglobulin Fc domain (e.g., Fc-knob) at the C-terminus of the heavy chain of the anti-TAA Fab antibody.

[0192] [Figure 5-2]Figure 5O shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA single domain antibody fused to the N-terminus of the other of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole).

[0193] Figure 5P shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole).

[0194] Figure 5Q shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole), (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob).

[0195] Figure 5R shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole), (b) a cytokine peptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob).

[0196] Figure 5S shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-knob), (b) a cytokine peptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole).

[0197] Figure 5T shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, (b) an anti-TAA Fab antibody fused to the N-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., the Fc-hole) at the C-terminus of the heavy chain fragment of the anti-TAA Fab antibody, and (c) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the above anti-cytokine / anti-TAA two-in-one Fab antibody.

[0198] Figure 5U shows an immunoconjugate containing (a) an anti-cytokine / anti-TAA two-in-one Fab antibody fused to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) at the C-terminus of the heavy chain fragment of the anti-cytokine / anti-TAA two-in-one Fab antibody, and (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody.

[0199] [Figure 6-1] Figure 6A shows the homogeneity of an isotype control antibody (DP47GS), an immunoconjugate molecule having configuration 2 (FB-225), and naked cytokine (Knob-IL2hex) by HPLC using a TOSH SW3000 column. As shown in the figure, homogeneity was significantly improved when comparing the naked cytokine Knob-IL2hex to an immunoconjugate containing a cytokine-stabilized IL-2-binding antibody (FB-255).

[0200] [Figure 6-2] Figure 6B shows the thermal stability of the control antibody (DP47GS), the immunoconjugate molecule with configuration 2 (FB-FB225), and the naked cytokine (Knob-IL2hex) as measured by differential scanning fluorimetry. The peak at 53 °C indicates significant right-shifted denaturation of IL-2hex, indicating that IL-2 was stabilized by the two-in-one masking antibody (FB-225) in the form of an immunocytokine molecule.

[0201] [Figure 6-3] Figure 6C shows the accelerated stability of the IL-2-containing immunoconjugate described herein, as measured using size exclusion chromatography (SEC). As shown in the figure, the protein remained stable after storage at 40°C for 4 weeks or after five freeze-thaw cycles.

[0202] [Figure 7]Figure 7A shows the pharmacokinetics of the naked cytokine (Knob-IL2hex) control and immunoconjugate molecules with configuration 2 (FB-476) and configuration 20 (FB-559), respectively, upon administration to mice as a single dose at various dosages. Protein concentrations were determined by anti-human Fc ELISA.

[0203] Figure 7B is a schematic representation of the immunocytokine FB-476 in configuration 2 shown in Figure 5C. FB-476 has a K D It contains the anti-cytokine / anti-hFAP two-in-one Fab antibody D047, which has an affinity for IL2hex.

[0204] Figure 7C is a schematic representation of the immunocytokine FB-559 in configuration 20 shown in Figure 5U. FB-559 has a K of approximately 400 nM. D The antibody contains the anti-cytokine / anti-hFAP two-in-one Fab antibody D029 mutant with an affinity for IL2hex of 1000kJ / 1000kcal.

[0205] [Figure 8] Figure 8A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates having Configuration 1 (circles) or Configuration 2 (upper triangle; lower triangle; diamond; and left triangle), respectively, as shown in Figures 6B and 6C. Assays performed in the presence of naked IL-2 (squares) were included as a positive control. The X-axis shows the concentration (pM) of IL-2 or IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL2. Figure 8B is a schematic representation of an immunoconjugate molecule of Configuration 1 according to the present disclosure. Figure 8C is a schematic representation of an immunoconjugate molecule of Configuration 2 according to the present disclosure.

[0206] [Figure 9]Figure 9A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates having Configuration 1 (squares), Configuration 2 (circles), or Configuration 4 (triangles), as shown in Figures 7B, 7C, and 7D, respectively. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL2. Figure 9B is a schematic diagram of an immunoconjugate molecule of Configuration 1 according to the present disclosure. Figure 9C is a schematic diagram of an immunoconjugate molecule of Configuration 2 according to the present disclosure. Figure 9D is a schematic diagram of an immunoconjugate molecule of Configuration 4 according to the present disclosure.

[0207] [Figure 10] Figure 10A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates with configuration 1 (white square) or configuration 2 (white square with cross; blue square; pink square, red square), as shown in Figures 8B and 8C, respectively. Assays were performed in the presence (pink square, red square) or absence (white square, white square with cross; blue square) of soluble human fibroblast activation protein (hFAP). Assays performed in the presence of naked IL-2 (black square) were included as a positive control; assays performed without any immunoconjugate molecule but in the presence of soluble FAP (white square with dashed line) were included as a negative control. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 10B is a schematic diagram of an immunoconjugate molecule of Configuration 1 according to the present disclosure. Figure 10C is a schematic diagram of an immunoconjugate molecule of Configuration 2 according to the present disclosure.

[0208] [Figure 11-1]Figure 11A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates having Configuration 1 (squares) or Configuration 3 (triangles; circles), as shown in Figures 9B and 9C, respectively. Assays were performed in the presence (triangles) or absence (squares, circles) of cells expressing human fibroblast activation protein (hFAP) on the cell surface. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 11B is a schematic diagram of an immunoconjugate molecule of Configuration 1 according to the present disclosure. Figure 11C is a schematic diagram of an immunoconjugate molecule of Configuration 3 according to the present disclosure.

[0209] [Figure 11-2] Figure 11D shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates with configuration 1 (squares) or configuration 3 (triangles; circles), as shown in Figures 9B and 9C, respectively. Assays were performed with (blue triangles, red triangles, hexagons of sizes 1-4) or without (squares, circles) cells expressing human fibroblast activation protein (hFAP) on the cell surface, and with (red triangles, hexagons of sizes 1-4) or without (squares, circles) soluble FAP molecules. The X-axis shows the concentration of IL-2-containing immunoconjugates (pM); the Y-axis shows the absorbance at 635 nm (A635), determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2.

[0210] [Figure 11-3]Figure 11E shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates with configuration 1 (squares) or configuration 3 (triangles; circles), as shown in Figures 9B and 9C, respectively. Assays were performed with (lower triangle, diamond, pentagon, hexagon) or without (square, circle, upper triangle) cells expressing human fibroblast activation protein (hFAP) on the cell surface, and with (diamond, pentagon, hexagon) or without (square, circle, upper triangle, lower triangle) soluble antibody. The X-axis represents the concentration of IL-2-containing immunoconjugate (pM); the Y-axis represents the absorbance at 635 nm (A635), determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2.

[0211] [Figure 12] Figure 12A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates having Configuration 1 (black squares; white squares) or Configuration 2 (black triangles; white triangles), respectively, as shown in Figures 10B and 10C. Assays were performed in the presence of unmodified HEK293T cells (black squares, white squares, white triangles) or HEK293T cells expressing human fibroblast activation protein (hFAP) on the cell surface (black triangles). The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 12B is a schematic diagram of an immunoconjugate molecule of Configuration 1 according to the present disclosure. FIG. 12C is a schematic representation of an immunoconjugate molecule of Configuration 2 according to the present disclosure.

[0212] [Figure 13]Figure 13A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of an IL-2-containing immunoconjugate having Configuration 3, as shown in Figure 13B. Assays were performed in the presence (solid line, open circles, and open triangles) or absence (solid line, closed circles, and closed triangles) of cells expressing human fibroblast activation protein (hFAP) on the cell surface. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Both immunoconjugate molecules tested (FB-387) and (FB-392) were Configuration 3, containing the same two-in-one Fab D029. The anchoring moiety of FB-387 is scFv5, which has a KD for hFAP of approximately 5 nM and binds to a different epitope on hFAP than D029; the anchoring moiety of FB-392 is scFv70, which has a KD for hFAP of approximately 1 M and binds to the same epitope on hFAP as D029. Both molecules showed similar activity in the presence or absence of hFAP-expressing cells.

[0213] FIG. 13B shows a schematic representation of an immunoconjugate molecule of Configuration 3 according to the present disclosure.

[0214] [Figure 14] 14 is a schematic representation of soluble FAP-induced unmasking of IL2 contained in an immunoconjugate molecule of the present disclosure. The simultaneous binding of two FAP-binding moieties (anchoring moiety and two-in-one masking moiety) allows the cytokine peptide to dissociate from the masking moiety and become available to bind to 5UTZ, the human IL-2 / Fab complex shown in the figure.

[0215] [Figure 15] FIG. 15 shows biolayer interferometry (BLI) binding curves of immobilized 5UTZ to demasked IL2hex in four immunoconjugate molecules FB-604, FB-675, FB-676, and FB-626.

[0216] [Figure 16] Figure 16 shows biolayer interferometry (BLI) binding curves of immobilized 5UTZ molecules to soluble Fc-hFAP and Knob-IL2hex.

[0217] [Figure 17] Figure 17A shows the biolayer interferometry (BLI) curve of the immunoconjugate molecule FB-604, which was able to bind to immobilized 5UTZ molecules in the presence of soluble Fc-hFAP, but not in the absence of soluble Fc-hFAP.

[0218] Figure 17B is a schematic representation of the immunoconjugate molecule FB-604 in Configuration 2. The two-in-one antibody in FB-604 has a K of approximately 1.53 nM. D to FAP with a K value of approximately 1.59 μM D Bind to IL2hex by value.

[0219] [Figure 18] Figure 18A shows the biolayer interferometry (BLI) curve of the immunoconjugate molecule FB-675, which was able to bind to immobilized 5UTZ molecules in the presence of soluble Fc-hFAP, but not in the absence of soluble Fc-hFAP.

[0220] Figure 18B is a schematic representation of the immunoconjugate molecule FB-675 in Configuration 3. The two-in-one antibody in FB-675 has a K of approximately 3.66 nM. D value to FAP, and a K of approximately 217 nM D The anchoring moiety in FB-675 binds to IL2hex with a K of approximately 5 nM. D binds to FAP.

[0221] [Figure 19]Figure 19A shows the biolayer interferometry (BLI) curve of the immunoconjugate molecule FB-676, which was able to bind to immobilized 5UTZ molecules in the presence of soluble Fc-hFAP, but not in the absence of soluble Fc-hFAP.

[0222] Figure 19B is a schematic representation of the immunoconjugate molecule FB-676 in Configuration 3. The two-in-one antibody in FB-675 has a K of approximately 1.53 nM. D to FAP, and a K of approximately 1.59 μM D The anchoring moiety binds to IL2hex with a K of approximately 5 nM. D binds to FAP.

[0223] [Figure 20] Figure 20A shows the biolayer interferometry (BLI) curves of the immunoconjugate molecule FB-626, which was unable to bind to immobilized 5UTZ molecules either in the presence or absence of soluble Fc-hFAP.

[0224] Figure 20B is a schematic representation of the immunoconjugate molecule FB-626 of configuration 14. The two-in-one antibody in FB-626 has a K greater than about 5 μM. D to FAP, and a K of approximately 237 μM D It binds to IL2hex.

[0225] [Figure 21]Figure 21A shows IL-2 activity measured using an IL-2 reporter cell system in the presence of IL-2-containing immunoconjugates having Configuration 1 (squares) or Configuration 3 (black circles, black triangles, open circles, open triangles), as shown in Figures 21B and 21C. Assays were performed with (open circles, open triangles) or without (squares, black circles, black triangles) HEK293T cells expressing human fibroblast activation protein (hFAP) on the cell surface. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 21B is a schematic diagram of an immunoconjugate molecule of Configuration 1 according to the present disclosure. Figure 21C is a schematic diagram of an immunoconjugate molecule of Configuration 3 according to the present disclosure.

[0226] [Figure 22] Figure 22A shows IL-2 activity measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells HEK293T-hFAP-E5. Both tested immunoconjugate molecules had configuration 3, shown in Figure 22B, and contained the same anchor moiety with scFv872-5. The two tested immunoconjugate molecules had different masking moieties containing two-in-one antibodies D001 and D002, respectively. As shown in the figure, both immunoconjugate molecules had similar masking effects on cytokines in the absence of hFAP-expressing cells. Furthermore, both immunoconjugate molecules were able to unmask and activate cytokine activity in the presence of hFAP-expressing cells.

[0227] FIG. 22B is a schematic representation of an immunoconjugate molecule of Configuration 3 according to the present disclosure.

[0228] [Figure 23]Figure 23A shows IL-2 activity measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells HEK293T-hFAP-E5. Both tested immunoconjugate molecules had the same anchoring moiety, configuration 3, shown in Figure 23B, including scFv872-59. The two tested immunoconjugate molecules had different masking moieties, containing the two-in-one antibodies D001 and D002, respectively. As shown in the figure, both immunoconjugate molecules had similar masking effects on cytokines in the absence of hFAP-expressing cells. Furthermore, both immunoconjugate molecules were able to unmask and activate cytokine activity in the presence of hFAP-expressing cells.

[0229] FIG. 23B is a schematic representation of an immunoconjugate molecule of Configuration 3 according to the present disclosure.

[0230] [Figure 24] Figure 24A shows IL-2 activity measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells HEK293T-hFAP-E5. Both tested immunoconjugate molecules had the same anchoring moiety, configuration 3, shown in Figure 24B, including scFv872-70. The two tested immunoconjugate molecules had different masking moieties, containing the two-in-one antibodies D001 and D002, respectively. As shown in the figure, both immunoconjugate molecules had similar masking effects on cytokines in the absence of hFAP-expressing cells. Furthermore, both immunoconjugate molecules were able to unmask and activate cytokine activity in the presence of hFAP-expressing cells.

[0231] FIG. 24B is a schematic representation of an immunoconjugate molecule of Configuration 3 according to the present disclosure.

[0232] [Figure 25]Figure 25A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates having Configuration 1 (circles, squares) or Configuration 5 (white diamonds, black diamonds), as shown in Figures 12B and 12C, respectively. Assays were performed with (black diamonds) or without (squares, circles, white diamonds) HEK293T cells expressing human fibroblast activation protein (hFAP) on the cell surface. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 25B is a schematic diagram of an immunoconjugate molecule of Configuration 1 according to the present disclosure. Figure 25C is a schematic diagram of an immunoconjugate molecule of Configuration 5 according to the present disclosure.

[0233] [Figure 26-1] Figure 26A shows IL-2 activity measured using an IL-2 reporter cell line in the presence of IL-2-containing immunoconjugates with configuration 1 (circles, squares) or configuration 6 (open diamonds, open lower triangles, open left triangles, black diamonds, black lower triangles, black left triangles), as shown in Figures 13B and 13C, respectively. Assays were performed with (open diamonds, open lower triangles, open left triangles) or without (squares, circles, black diamonds, black lower triangles, black left triangles) HEK293T cells expressing human fibroblast activation protein (hFAP) on the cell surface. The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the absorbance at 635 nm (A635), determined using a TECAN plate reader, which reflected secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 26B is a schematic representation of immunoconjugate molecules of Configuration 1 according to the present disclosure. Figure 26C is a schematic representation of immunoconjugate molecules of Configuration 6 according to the present disclosure. Figure 26D is a bar graph showing the quantified EC50 (pM) values ​​for the assays in the study. [Figure 26-2] Same as above.

[0234] [Figure 27] Figure 27A shows the IL-2 activity measured using an IL-2 reporter cell system for two immunoconjugate molecules, FB-676 and FB-707, in the presence or absence of FAP-expressing cells, HEK293T-hFAP-E5. The EC50 was approximately 14 nM for shielded FB-676 and approximately 40 pM for unshielded FB-676; the EC50 was approximately 12 nM for shielded FB-707 and approximately 11 pM for unshielded FB-707. The potency of IL-2 increased approximately 700-1000-fold in the presence of FAP-expressing cells compared to the absence of FAP-expressing cells.

[0235] Figure 27B is a schematic representation of the immunoconjugate molecule FB-707 of configuration 15. The two-in-one antibody in FB-707 has a K of approximately 1.53 nM. D to FAP, and a K of approximately 1.59 μM D The anchoring moiety binds to IL2hex with a K of approximately 5 nM. D binds to FAP.

[0236] Figure 27C is a schematic representation of the immunoconjugate molecule FB-676 in Configuration 3. The two-in-one antibody in FB-675 has a K of approximately 1.53 nM. D to FAP, and a K of approximately 1.59 μM D The anchoring moiety binds to IL2hex with a K of approximately 50 nM. D binds to FAP.

[0237] [Figure 28]Figure 28A shows the activation of human CD4+ T cells by immunoconjugate molecules of the present disclosure, as measured using a pSTAT5 staining assay. The ability of immunoconjugate molecules FB-604, FB-674, FB-675, and FB-676 to stimulate preactivated human CD4+ cells was measured in the presence or absence of 200 nM Fc-hFAP. As shown in the figure, IL2hex potency was increased approximately 2-fold by the immunoconjugate molecule FB-604, which does not have an anchoring moiety, and approximately 10-fold for all other tested immunoconjugate molecules that have anchoring moieties.

[0238] Figure 28B shows the activation of human CD4+ T cells by immunoconjugate molecules of the present disclosure, as measured using a pSTAT5 staining assay. The ability of immunoconjugate molecules FB-801, FB-794, FB-818, and FB-834 to stimulate preactivated human CD4+ cells was measured in the presence or absence of 200 nM Fc-hFAP. As shown in the figure, IL2hex potency was increased approximately 30-fold for all tested immunoconjugate molecules with anchoring moieties.

[0239] [Figure 29-1] Figure 29A shows the activation of human CD4+ T cells by immunoconjugate molecules of the present disclosure as measured using a pSTAT5 staining assay. The ability of immunoconjugate molecules FB-611, FB-610, FB-609, FB-608, FB-607, FB-601, FB-600, FB-599, FB-598, FB-676, FB-675, FB-674, and FB-604 to stimulate preactivated human CD4+ cells was measured in the presence or absence of 200 nM Fc-hFAP.

[0240] [Figure 29-2] FIG. 29B shows the quantification of the EC50 values ​​measured by the assay in FIG. QA.

[0241] [Figure 30]FIG. 30 shows the acute toxicity of Knob-IL2hex to C57BL / 6J and CB-17 SCID mice as measured by mortality (left) and weight loss (right).

[0242] [Figure 31-1] Figure 31A shows purified immunoconjugate molecules on non-reducing and reducing SDS-PAGE gels for four protein samples: control (Knob-IL2hex, MW = 66.8 kDa), FB-439 (MW = 92.3 kDa), FB-449 (MW = 120 kDa), and FB-476 (MW = 116 kDa).

[0243] [Figure 31-2] Figures 31B-31D show the potency of immunoconjugate molecules FB-439, FB-449, and FB-476 as measured by the CTLL2 proliferation assay, the NK92 proliferation assay, and the HEK Blue IL2 activation assay, respectively. [Figure 31-3] Same as above. [Figure 31-4] Same as above.

[0244] [Figure 31-5] Figure 31E shows human CD4+ T cell proliferation by immunoconjugate molecules of the present disclosure as measured Alarm Blue fluorescence. The ability of the immunoconjugate molecule FB-794 to stimulate preactivated human CD4+ cells was measured by co-culturing them with 40k fixed ExpiCHO cells with or without hFAP receptors on their surface in the presence or absence of 200 nM Fc-hFAP.

[0245] [Figure 32-1] FIG. 32A shows measurements of mortality (left) and weight loss (right) in mice administered the immunoconjugate molecules: control (Knob-IL2hex), FB-439, FB-449, FB-476.

[0246] [Figure 32-2]FIG. 32B shows measurements of weight loss in mice administered the immunoconjugate molecule sKnob-IL2hex (control), FB-439, FB-476, or PBS (control).

[0247] [Figure 33-1] FIG. 33A is a 3D representation of the IL-2 molecule binding to the IL-2R α, β, and γ subunits (PDB: 2ERJ).

[0248] [Figure 33-2] Figures 33B and 33C show the binding kinetics of a two-in-one antibody (B10) to IL-2 and FAP, respectively, compared with two other IL-2 antibodies (i.e., 5UTZ, which blocks IL-2 binding to IL-2Rβ (CD122), and NARA1, which blocks IL-2 binding to IL-2Rα (CD25). B10 binds to IL-2 at an epitope that overlaps with NARA1, but not to 5UTZ. [Figure 33-3] Same as above.

[0249] [Figure 34-1]Figure 34A shows the IL-2 activity of the immunoconjugate molecule FB-1097 measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells. The immunoconjugate tested had configuration 15, shown in Figure 34B. The immunoconjugate contained point mutations in IL-2 (T3A, K35E, F42A, Y45A, L72G, C125S). The immunoconjugate also contained a variant of D029 Fab as the masking moiety and a variant of scFv 872-5 as the anchoring moiety. An IL-2 Fc fusion protein with configuration 1, containing both wild-type IL-2 (black circles) and mutant IL-2hex (squares), was included as a control. Assays were performed in the presence of cells that expressed human fibroblast activation protein (hFAP) on the cell surface (HEK293T-hFAP-E5; lower triangles) or in the presence of cells that did not express FAP (HEK293T upper triangles). The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the activity of the immunoconjugate using a TECAN plate reader, which reflects secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2. Figure 34C shows tumor size and body weight of the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), CTRL-IL2hex, 55 μg of FB-1097, or 220 μg of FB1097.

[0250] [Figure 34-2] Figure 34D shows the systemic expansion of CD3+CD4+, CD3+CD8+, and NK cells in the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), 12.5 μg of CTRL-IL2WT, 12.5 μg of CTRL-IL2hex, or 220 μg of FB-1097. Figure 34E shows the lung weight of the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), 12.5 μg of CTRL-IL2WT, 12.5 μg of CTRL-IL2hex, or 220 μg of FB-1097.

[0251] [Figure 35-1] Figure 35A shows the IL-2 activity of immunoconjugate molecule #1112 measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells. The immunoconjugate tested had configuration 14, shown in Figure 35B. The immunoconjugate contained point mutations in IL-2 (T3A, K35E, F42A, C125S). The immunoconjugate also contained D029H and D029L masking moieties and the anchoring moiety VHH-E33. An IL-2 Fc fusion protein with configuration 1, containing both wild-type IL-2 (circles) and mutant IL-2hex (black squares), was included as a control. Assays were performed in the presence of cells that expressed human fibroblast activation protein (hFAP) on the cell surface (HEK293T-hFAP-E5; open squares) or cells that did not express FAP (HEK293T triangles). The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the activity of the immunoconjugate using a TECAN plate reader, which reflects secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL-2.

[0252] [Figure 35-2] FIG. 35C shows tumor size and body weight of the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), 25 μg of CTRL-IL2 F42A, 55 μg of FB-1112, or 220 μg of FB-1112.

[0253] Figure 35D shows the systemic expansion of CD3+CD4+, CD3+CD8+, and NK cells in the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), 12.5 μg of CTRL-IL2hex, 55 μg of FB-1112, or 220 μg of FB-1112.

[0254] FIG. 35E shows lung weights of the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), 12.5 μg of CTRL-IL2hex, 55 μg of FB-1112, or 220 μg of FB-1112.

[0255] [Figure 36-1] Figure 36A shows the IL-2 activity of immunoconjugate molecule #1150 measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells. The immunoconjugate tested had configuration 14, as shown in Figure 36B. The immunoconjugate also contained a Fab derived from antibody B10 as a masking moiety and an anchoring moiety, VHH-E33. An IL-2 Fc fusion protein with configuration 1, carrying both wild-type IL-2 (black circles) and mutant IL-2hex (squares), was included as a control. Assays were performed in the presence of cells that expressed human fibroblast activation protein (hFAP) on the cell surface (B-MC38-FAP; open upper triangles) or did not express FAP (MC38 black upper triangles). The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the activity of the immunoconjugate using a TECAN plate reader, which reflects secreted embryonic alkaline phosphatase (SEAP) levels and response to IL-2.

[0256] [Figure 36-2] Figures 36C-D show tumor size (Figure 36C), survival rate (Figure 36D), and body weight change (Figure 36E) measured in the MC38-FAP tumor model in C57BL / 6 mice administered vehicle (PBS), 12.5 μg of CTRL-IL2D20T, or 55 μg of FB-1150.

[0257] [Figure 37-1]Figure 37A shows the IL-2 activity of immunoconjugate molecule #1125 measured using an IL-2 reporter cell system in the presence or absence of FAP-expressing cells. The immunoconjugate tested had configuration 14, as shown in Figure 37B. The immunoconjugate also contained a Fab derived from antibody B10 as a masking moiety and the anchoring moiety scFv872-5. An IL-2 Fc fusion protein with configuration 1, carrying both wild-type IL-2 (black circles) and mutant IL-2hex (white circles), was included as a control. Assays were performed in the presence of cells that expressed human fibroblast activation protein (hFAP) on the cell surface (B-MC38-FAP; white upper triangles) or did not express FAP (MC38 black upper triangles). The X-axis shows the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis shows the activity of the immunoconjugate using a TECAN plate reader, which reflects secreted embryonic alkaline phosphatase (SEAP) levels and response to IL-2.

[0258] [Figure 37-2] FIG. 37C shows tumor volume in the MC38 tumor model in C57BL / 6 mice administered PBS, 12.5 μg of CTRL D20T, or 220 μg of FB-1125.

[0259] FIG. 37D shows tumor volume of the MC38-FAP tumor model in C57BL / 6 mice administered 12.5 μg of CTRL D20T, 55 μg of FB-1125, or 55 μg of FB-1125 and 100 μg of si-4B9.

[0260] [Figure 38-1]Figures 38A and 38B show IL-2 activity measured in various cells using an IL-2 reporter cell system by screening immunoconjugate molecule A and corresponding molecular configurations. Immunoconjugate molecule A contains an IL-2 moiety, a two-in-one masking moiety capable of binding to IL-2 and EpCAM (Fab derived from antibody FL78), and an anti-EpCAM anchoring moiety (scFv derived from MOC31). The assay was performed in the presence of HEK293T EpCAM (high) cells, which express EpCAM on the cell surface. HEK293T cells did not express EpCAM. Molecule A has the same scaffold as configuration 15. The X-axis indicates the concentration (pM) of the IL-2-containing immunoconjugate; the Y-axis indicates the absorbance at 635 nm (A635) determined using a TECAN plate reader, which reflects secreted embryonic alkaline phosphatase (SEAP) levels and the response to IL2.

[0261] [Figure 38-2] FIG. 38C shows biolayer interferometry (BLI) binding curves of immobilized EpCAM and mutant IL2 IL-2hex(K35E) molecules to immunoconjugate molecule A shown in FIG. 38A. DETAILED DESCRIPTION OF THE INVENTION

[0262] 5. Detailed Description The present disclosure provides immunoconjugate molecules comprising cytokine polypeptides. In certain embodiments, the present disclosure also provides polynucleotides and vectors comprising sequences encoding such immunoconjugate molecules, as well as compositions, reagents, and kits comprising such immunoconjugate molecules. In a related aspect, methods are also provided herein for using immunoconjugate molecules according to the present disclosure to deliver and / or activate cytokine activity at a target site, or to reduce toxicity and / or other side effects associated with systemic exposure to cytokine activity in a subject.

[0263] The present disclosure also provides, in certain embodiments, peptides or polypeptides, such as antibodies or antigen-binding fragments thereof, that can form part of such immunoconjugate molecules of the present disclosure. In specific embodiments, provided herein are binding proteins comprising antibodies or fragments thereof that bind to fibrosis activating proteins (FAPs). In specific embodiments, provided herein are bispecific binding proteins comprising two-in-one antibodies or fragments thereof that bind to both FAPs and interleukin-2 (IL-2). 5.1 General techniques

[0264] The techniques and procedures described or referenced herein are those generally well understood and / or commonly employed by those of skill in the art using conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); Phage Display in Biotechnology and Drug Discovery (Sidhu and Geyer eds., 2nd ed. 2005); Phage Display: a Laboratory Manual (Barbas et al. eds., 2004); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. This includes widely used methodologies such as those described in (2010). 5.2 Terminology

[0265] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following explanations of terms shall apply, and whenever appropriate, terms used in the singular shall also include the plural, and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. If any explanation of a given term conflicts with any document incorporated herein by reference, the explanation of the term set forth below shall control.

[0266] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0267] Unless otherwise indicated, the terms "oligonucleotide" and "nucleic acid" are used interchangeably and are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxy orientation, respectively. Thus, the codon at the 5' end of an oligonucleotide generally corresponds to the N-terminal amino acid residue that will be incorporated into the translated protein or peptide product. Similarly, the codon at the 3' end of an oligonucleotide generally corresponds to the C-terminal amino acid residue that will be incorporated into the translated protein or peptide product. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art.

[0268] The term "interleukin-2" or "IL-2," as used herein, unless otherwise indicated, refers to any native IL-2 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 and any form of IL-2 that results from processing in cells. The term also encompasses naturally occurring variants of IL-2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is: [ka] Unprocessed human IL-2 additionally contains an N-terminal 20 amino acid signal peptide (underlined, not present in the mature IL-2 molecule) and has the sequence shown below: [ka] It has.

[0269] Without being bound by theory, it is contemplated that the IL-2 polypeptide binds to the IL-2 receptor (IL-2R) at the α, β, and / or γ subunits of the IL-2R receptor complex. Furthermore, the region of IL-2 involved in binding to IL-2Rα (CD-25) is P34 (3.2 Å). 2 ), K35(37Å 2 ), R38(130Å 2 ), T41(25Å 2 ), F42(95Å 2 ), K43(61Å 2 ), F44(4Å 2 ), Y45(90Å 2 ), E61(67Å 2 ), E62(15Å 2 ), K64(46Å 2 ), P65(46Å 2 ), E68(78Å 2 ), V69(6Å 2 ), N71(3Å 2 ), L72(49Å 2 ), Q74(43Å2 ), Y107(35Å 2 ), D109(3Å 2 The region of IL-2 involved in binding to IL-2Rβ (CD122) contains L12 (40 Å 2 ), Q13(29Å 2 ), E15(37Å 2 ), H16(89Å 2 ), L19(68Å 2 ), D20(24Å 2 ), M23(33Å 2 ), R81(51Å 2 ), D84(57Å 2 ), D87(16Å 2 ), N88(62Å 2 ), V91(85Å 2 ), I92(34Å 2 ), E95(45Å 2 ), and that the region of IL-2 involved in binding to IL-2rγ (CD-132) contains Q11 (9Å 2 ), L12(3Å 2 ), E15(41Å 2 ), L18(19Å 2 ), L19(12Å 2 ), Q22(28Å 2 ), K48(8Å 2 ), T51(1Å 2 ), E110(26Å 2 ), N119(59Å 2 ), R120(5Å 2 ), I122(5Å 2 ), T123(59Å 2 ), Q126(82Å 2 ), S127(32Å 2 ), I129(40Å 2 ), S130(55Å 2 ), T131(<1Å 2 ), T133(29Å 2 ), where the number in parentheses is the calculated buried surface area from the IL-2 receptor protein complex using Protein Data Bank ID 2B5I.

[0270] The term "IL-2 mutant" or "mutant IL-2 polypeptide," as used herein, is intended to encompass any mutant form of the various forms of the IL-2 molecule, including full-length IL-2, truncated forms of IL-2, and forms in which the IL-2 polypeptide contains one or more amino acid mutations in its sequence. "Full-length," when used in reference to IL-2, is intended to mean the mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, e.g., SEQ ID NO: 1). Various forms of IL-2 mutants are characterized as having at least one amino acid mutation that affects the interaction of IL-2 with CD25. This mutation may include substitution, deletion, truncation, or modification of the wild-type amino acid residue normally located at that position. Unless otherwise indicated, IL-2 mutants may be referred to herein as IL-2 mutant peptide sequences, IL-2 mutant polypeptides, IL-2 mutant proteins, or IL-2 mutant analogs. The designations of various forms of IL-2 are made herein with reference to the sequence shown in SEQ ID NO: 1. Various designations may be used herein to designate the same mutation. For example, a mutation from phenylalanine to alanine at position 42 is designated 42A, A42, A 42 , F42A, or Phe42Ala. The designation "IL-2hex" refers to a mutant form of human IL-2 shown below, which contains the ΔA1 / T3A / F42A / Y45A / L72G / C125S mutations in the human IL-2 sequence (amino acid substitutions are underlined and bold): [ka] The numbering of the mutated amino acid residue positions is according to the wild-type human IL-2 sequence (SEQ ID NO: 1). Without being bound by theory, it is contemplated that the mutation ΔA1 removes the N-terminal residue of the mature form of IL-2; the mutation T3A removes a potential glycosylation site; the F42A / Y45A / L72G mutations reduce binding of IL-2 to CD25; and the C125S mutation removes an unpaired cysteine ​​in IL-2.

[0271] Without being bound by theory, it is contemplated that mutations in the region of the IL-2 polypeptide responsible for IL-2 interaction with one IL-2R subunit may affect IL-2 binding to that IL-2R subunit, while not affecting IL-2R binding to another IL-2R subunit. For example, various IL-2 mutations, including but not limited to, K35E, R38A, R38D, R38E, F42A, F42K, K43E, Y45A, E61R, E62A, L72G, or combinations thereof, are known to negatively affect the binding of IL-2 to IL-2Rα (CD25). For example, a single F42A mutation has been demonstrated to reduce binding of IL-2 to IL-2α by approximately 100-fold, while combinations of (a) F42A / Y45A / L72G, (b) R38D / K43E / E61R, or (c) R38A / F42A / Y45A / E62A have been demonstrated to completely abolish binding of IL-2 to IL-2α. A variety of IL-2 mutations are known to negatively affect binding of IL-2 to IL-2Rβ (CD122), including, but not limited to, D20T, D20G, D20A, H16E, H16R, H16A, N88D, N88S, N88R, V91G, V91A, V91R, V91S, or combinations thereof. Various IL-2 mutations are known to affect the binding of IL-2 to IL-2Rγ (CD132), including, but not limited to, L18R, Q22E, T123A, and Q126X, where X=H, M, K, R, E, S, G, A, C, D, I, or T, I129V, S130A, S130R, or a combination thereof. Combinations of IL-2 mutations that affect binding to IL-2Rγ have been used to create agonists and inhibitors of IL-2 signaling. For example, it has been demonstrated that the Q126T mutation combined with the Q74H / L80F / R81D / L85V / I92F mutation enhances the binding of IL-2 to IL-2Rγ and can act as a partial agonist of IL-2 receptor signaling.For another example, it has been demonstrated that the L18R / Q22E / Q126T / S130R mutation combination in IL-2 abolishes IL-2 signaling and may play a role in inhibiting wild-type IL-2 signaling.

[0272] Additional exemplary IL-2 mutants that can be used in conjunction with the present disclosure also include: [ka] IL2 C125S (residues 1-153, no signal peptide, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2 C125A (residues 1-153, no signal peptide, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-F42A / Y45A / L72G / C125A (residues 1 to 153, no signal peptide, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-R38A / F42A / Y45A / E62A / C125S (residues 1 to 153, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-T3A / R38E / F42A / C125S (residues 1-153, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-T3A / R38E / Y45A / C125S (residues 1-153, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-T3A / R38E / L72G / C125S (residues 1-153, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-ΔA2 / T3A / F42A / Y45A / L72G / C125S (residues 2–153, no signal peptide “hex”, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-ΔA2 / T3A / K35E / F42A / Y45A / L72G / C125S (residues 2–153, without signal peptide “hex / K35E”, amino acid substitutions are underlined and bolded), with the sequence: [ka] IL2-T3A / K35E / F42A / Y45A / L72G / C125S (residues 1 to 153, no signal peptide, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-T3A / K35E / F42A / C125S (residues 1 to 153, no signal peptide, amino acid substitutions are underlined and bolded), which has the sequence: [ka] IL2-T3A / D20T / K35E / C125S (residues 1-153, no signal peptide, amino acid substitutions underlined and bolded), having the sequence [ka] IL2-T3A / H16A / K35E / C125S (residues 1 to 153, no signal peptide, amino acid substitutions are underlined and bolded), which has the sequence:

[0273] Additional mutant IL-2 polypeptides that can be used in conjunction with the present disclosure include, for example, those described in U.S. Pat. Nos. 10,184,009 and 5,229,109, and International Patent Publication No. WO2012107417A1, the disclosures of each of which are incorporated herein by reference in their entireties.

[0274] As used herein, a "wild-type" form of IL-2 is a form of IL-2 that is otherwise identical to the mutant IL-2 polypeptide, except that the wild-type form has the wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if an IL-2 mutant is full-length IL-2 (i.e., IL-2 that is not fused or conjugated to any other molecule), the wild-type form of the mutant is full-length native IL-2. If an IL-2 mutant is a fusion between IL-2 and another polypeptide (e.g., an antibody chain) encoded downstream of IL-2, the wild-type form of the IL-2 mutant is IL-2 with the wild-type amino acid sequence fused to the same downstream polypeptide. Furthermore, if an IL-2 mutant is a truncated form of IL-2 (a mutated or altered sequence within the non-truncated portion of IL-2), the wild-type form of the IL-2 mutant is a similarly truncated IL-2 with the wild-type sequence. For purposes of comparing the IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants with the corresponding wild-type form of IL-2, the term wild-type encompasses forms of IL-2 that contain, as compared to naturally occurring native IL-2, one or more amino acid mutations that do not affect receptor binding of IL-2, such as, for example, a substitution of alanine for cysteine ​​at a position corresponding to residue 125 in human IL-2. In certain embodiments according to the invention, the wild-type IL-2 polypeptide to which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO:1.

[0275] The term "CD25" or "α subunit of the IL-2 receptor" or "IL-2Rα," as used herein, unless otherwise indicated, refers to any native CD25 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD25, and any form of CD25 that results from processing in cells. The term also encompasses naturally occurring variants of CD25, e.g., splice variants or allelic variants. In certain embodiments, the CD25 is human CD25. The amino acid sequence of an exemplary human CD25 (with signal sequence, underlined) is shown below: [ka] .

[0276] The term "CD122" or "β subunit of the IL-2 receptor" or "IL-2R β," as used herein, unless otherwise indicated, refers to any native CD122 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD122 and any form of CD122 that results from processing in cells. The term also encompasses naturally occurring variants of CD122, e.g., splice variants or allelic variants. In certain embodiments, the CD122 is human CD122. The amino acid sequence of an exemplary human CD122 is shown below: [ka] .

[0277] The term "CD132" or "γ subunit of the IL-2 receptor" or "IL-2Rγ," as used herein, unless otherwise indicated, refers to any native CD132 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD132 and any form of CD132 resulting from processing in cells. The term also encompasses naturally occurring variants of CD132, e.g., splice variants or allelic variants. In certain embodiments, the CD132 is human CD132. The amino acid sequence of an exemplary human CD132 (with signal sequence, underlined) is shown below: [ka] .

[0278] The term "high affinity IL-2 receptor" as used herein refers to the receptor γ subunit (common cytokine receptor γ subunit, γ c , or CD132), a receptor beta subunit (also known as CD122 or p70), and a receptor alpha subunit (also known as CD25 or p55), or a functional variant thereof. In contrast, the term "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only the gamma and beta subunits, without the alpha subunit, or a functional variant thereof (for a review, see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0279] The term "tumor-associated antigen" or "TAA," as used herein, refers to an antigen expressed by cancer cells or in the stroma of a solid tumor. TAAs can be proteins, nucleic acids, lipids, or other antigens. In certain embodiments, TAAs can be cell-surface-expressed TAAs. In the context of solid tumors, TAAs can be expressed in the stroma of a solid tumor mass. The term "stroma," as used herein, refers to components in a solid tumor mass other than cancer cells. For example, stroma can include fibroblasts, epithelial cells, other vascular components, or extracellular matrix components. As used herein, the term "stroma" does not include components of the immune system, such as immune cells (e.g., B cells, T cells, dendritic cells, macrophages, natural killer cells, etc.). Various TAAs are known in the art. Identifying TAAs can be performed using methods known in the art, for example, those disclosed in Zhang et al., Methods Mol. Biol., 520:1-10 (2009), the contents of which are incorporated herein by reference.

[0280] The term "fibroblast activation protein" or "FAP," as used herein, unless otherwise indicated, refers to any native FAP from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed FAPs and any form of FAP that results from processing in cells. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. The amino acid sequence of an exemplary human FAP is shown below: [ka] .

[0281] The term "tumor microenvironment" refers to any and all elements of a neoplastic environment that create a structural and / or functional environment for the neoplastic process to survive, grow, and / or spread. As a non-limiting example, a tumor microenvironment is comprised of cells, molecules, fibroblasts, extracellular matrix, and / or blood vessels surrounding and / or supplying one or more neoplastic cells, such as a solid tumor. In certain embodiments, the neoplastic disease is a solid tumor. Exemplary cells or tissues within the tumor microenvironment include, but are not limited to, tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells, macrophages, neutrophils, and other immune cells located in close proximity to the tumor. Exemplary cellular functions that influence the tumor microenvironment include, but are not limited to, cytokine and / or chemokine production, responses to cytokines, antigen processing and presentation of peptide antigens, modulation of leukocyte chemotaxis and migration, modulation of gene expression, complement activation, modulation of signal transduction pathways, cell-mediated cytotoxicity, cell-mediated immunity, humoral immune responses, and innate immune responses.

[0282] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or univalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and antibody fragments, as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies derived from other species, such as mice and rabbits. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides, which are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (approximately 50-70 kDa) and one light chain (approximately 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). In specific embodiments, a specific molecular antigen can be bound by an antibody provided herein, e.g., an IL-2 polypeptide, an IL-2 fragment, or an IL-2 epitope. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments such as IL-2-binding fragments), which refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments such as IL-2-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments (including, e.g., monospecific, bispecific, etc.), F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, minibodies, and single-domain antibodies (VHHs or nanobodies). In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules containing an antigen-binding domain or antigen-binding site that binds to an IL-2 antigen (e.g., one or more CDRs of an anti-IL-2 antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.

[0283] The term "monoclonal antibody," as used herein, refers to an antibody obtained from an individual antibody comprising a population of substantially homogeneous antibodies, e.g., a population that is identical except for possible naturally occurring mutations that may be present in minor amounts, with each monoclonal antibody typically recognizing a single epitope on an antigen. In a specific embodiment, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell, wherein the antibody binds to only one epitope, as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art. The term "monoclonal" is not limited to any particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., 1975, Nature 256:495, or may be produced using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002). Exemplary methods for producing monoclonal antibodies are provided in the Examples herein.

[0284] "Polyclonal antibody," as used herein, refers to an antibody population generated in an immunogenic response to a protein with many epitopes, and thus includes a variety of different antibodies directed against the same or different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (see, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002)).

[0285] An "antigen" is a predetermined antigen to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.

[0286] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that contains the amino acid residues (e.g., CDRs) that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen.

[0287] As used herein, a "bispecific antibody" refers to an antibody or antigen-binding fragment thereof that can bind to two different target antigens. As used herein, a "two-in-one antibody" refers to a bispecific antibody that can bind to two different target antigens via a single antigen-binding domain. In some embodiments, the target antigens compete with each other for binding to the single antigen-binding domain of the two-in-one antibody, such that when the two-in-one antibody binds to one target antigen, it dissociates from the other target antigen.

[0288] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds, e.g., a localized region on the surface of an antigen, such as an IL-2 polypeptide or IL-2 polypeptide fragment, which can bind to one or more antigen-binding regions of an antibody and has antigenic or immunogenic activity and can elicit an immune response in an animal, such as a mammal (e.g., a human). An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody binds, as determined by any method known in the art, including, for example, by immunoassay. An antigenic epitope is not necessarily immunogenic. Epitopes often consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural and charge characteristics. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by consecutive sequences of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but join together during the folding of the protein into its three-dimensional structure. Induced epitopes are formed when the three-dimensional structure of a protein is in an altered conformation, such as after activation or binding of another protein or ligand. Generally, an antigen has several or many different epitopes and can react with many different antibodies. In certain embodiments, an antigen (e.g., a FAP) can have more than one epitope that is recognized and bound by different anti-FAP antibodies. In certain embodiments, different anti-FAP antibodies compete with each other for binding to the same epitope on a FAP.

[0289] An antibody binds the "epitope," "essentially the same epitope," or "same epitope" of a reference antibody if the two antibodies recognize identical, overlapping, or adjacent epitopes in three-dimensional space. The most widely used rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competitive assay, which can be configured in several different formats, for example, using either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive, fluorescent, or enzyme label.

[0290] "Epitope mapping" is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. "Epitope binning" is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for identifying the epitope recognition characteristics of different antibodies using competitive assays combined with computer processes for clustering antibodies based on their epitope recognition characteristics and for identifying antibodies with distinct binding specificities.

[0291] The term "binds" or "binding" refers to an interaction between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope on a target molecule, such as IL-2, is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) association rate (k on) to the ratio (k off / k on ) is the dissociation constant K, which is inversely related to affinity D It is. K D The lower the value, the higher the affinity of the antibody. D The value of k will vary for different complexes of antibody and antigen. on and k off The dissociation constant K for the antibodies provided herein depends on both D can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at a binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants, such as multivalent IL-2, contacts an antibody containing multiple binding sites, the antibody's interaction with the antigen at one site increases the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity. The avidity of an antibody can be a better measure of its binding ability than the affinity of its individual binding sites. For example, high avidity can offset low affinity, as may be found for a pentameric IgM antibody, which has lower affinity than IgG, but the high avidity of IgM resulting from its multivalency allows it to bind to the antigen effectively.

[0292] The terms "antibody that specifically binds to an antigen," "antibody that specifically binds to an epitope," and similar terms are also used interchangeably herein and refer to an antibody that specifically binds to an antigen, or a fragment or epitope of an antigen. Antibodies that specifically bind to an antigen can be identified, for example, by immunoassay, Biacore®, or other techniques known to those skilled in the art. An antibody specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens, as determined using experimental techniques, for example, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least two times the background signal or noise and may be more than 10 times higher than background. For a discussion of antibody specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2nd ed. 1989). An antibody that "binds an antigen of interest (e.g., a target antigen such as IL-2)" is one that binds the antigen with sufficient affinity so that it is useful as a therapeutic agent targeting cells or tissues expressing the antigen and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to "non-target" proteins is less than about 10% of the antibody's binding to its specific target protein, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. With respect to antibody binding to a target molecule, the terms "specific binding," "specifically binds to," or "is specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is distinct from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target.In this case, specific binding is demonstrated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. The terms "specific binding," "specifically binding to," or "specific for" a specific polypeptide or epitope on a specific polypeptide target, as used herein, refer to binding when a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In certain embodiments, an antibody that binds to an antigen of the present disclosure has a dissociation constant (K) of less than or equal to 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. D )

[0293] The term "compete," when used in the context of antibodies (e.g., antibodies and binding proteins that bind to cell surface antigens and compete for the same epitope or binding site on a target), refers to competition as determined by an assay in which the antibody (or binding fragment) under study prevents or inhibits specific binding of a reference molecule (e.g., a reference ligand or reference antigen-binding protein, e.g., a reference antibody) to a common antigen (e.g., a FAP or fragment thereof). Many types of competitive binding assays can be used to determine whether a test antibody competes with a reference antibody for binding to an antigen (e.g., a human FAP). Examples of assays that can be used include solid-phase direct or indirect RIA, solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-53), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-19), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988)), solid-phase direct labeling RIA using I-125 label (see, e.g., Morel et al., 1988, Mol. Immunol. 25:7-15), and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen (e.g., IL-2) bound to a solid surface, or cells bearing either an unlabeled test antigen-binding protein (e.g., a test anti-IL-2 antibody) or a labeled reference antigen-binding protein (e.g., a reference anti-IL-2 antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess.Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and / or antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody so that steric hindrance of the antibody occurs. Further details regarding methods for determining competitive binding are described herein. Typically, when a competing antibody protein is present in excess, it inhibits the specific binding of the reference antibody to a common antigen by at least 30%, for example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0294] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 50 to 70 kDa, in which the amino-terminal portion contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion contains a constant region. The constant region can be one of five distinct types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the heavy chain constant region. Distinct heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to the five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, which contain the four IgG subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0295] The term "light chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 25 kDa, in which the amino-terminal portion contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Two distinct types exist, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

[0296] The terms "variable region," "variable domain," "V region," or "V domain" refer to the portion of an antibody light or heavy chain that is generally located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length in heavy chains and approximately 100-110 amino acids in length in light chains, and that is used for binding and specificity of each particular antibody to its specific antigen. The variable region of a heavy chain is sometimes referred to as "VH." The variable region of a light chain is sometimes referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary greatly in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the 110-amino acid span of the variable region. In fact, V regions consist of less variable (e.g., relatively invariant) sections of approximately 15-30 amino acids called framework regions (FRs) separated by shorter regions of higher variability (e.g., highly variable) called "hypervariable regions," each approximately 9-12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs, primarily adopting a β-sheet configuration, connected by three hypervariable regions, which form connecting loops and, in some cases, part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, together with the hypervariable regions from other chains, contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant region is not directly involved in binding the antibody to the antigen, but exhibits various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequences of the variable regions vary greatly between different antibodies. In a specific embodiment, the variable regions are human variable regions.

[0297] The terms "variable region residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a "standard" Kabat-numbered sequence at the region of homology. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0298] "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of the antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art, and are defined, for example, by Kabat as the most hypervariable region within the variable (V) domain of an antibody (Kabat et al., 1997, J. Biol. Chem. 252:6609-16; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR region sequences have also been structurally defined by Chothia as residues that are not part of a conserved β-sheet framework and therefore can adopt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The positions of CDRs within the variable regions of reference antibodies have been determined by comparison of multiple structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues in hypervariable regions varies in different antibodies, additional residues relative to the reference positions are conventionally numbered a, b, c, etc. next to the residue number in the reference variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is likewise well known to those of skill in the art.

[0299] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several hypervariable region descriptions are in use and are encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra). Chothia instead refers to the location of structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2d ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues from each of these hypervariable regions or CDRs are described below.

[0300] Recently, a universal numbering system, the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77), has been developed and widely adopted. IMGT is an integrated information system detailing immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) from humans and other vertebrates. Here, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and occurs in structures called loops, by using a numbering system that aligns variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in the grafting and replacement of CDR residues from one immunoglobulin species onto an acceptor framework, typically derived from a human antibody. The additional numbering system (AHon) was developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309: 657-70. Correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). In some embodiments, the CDRs are as defined by the IMGT numbering system. In other embodiments, the CDRs are as defined by the Kabat numbering system. In certain embodiments, the CDRs are as defined by the AbM numbering system. In other embodiments, the CDRs are as defined by the Chothia system. In still other embodiments, the CDRs are as defined by the Contact numbering system. [Table 16]

[0301] The hypervariable regions may include "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "HVR" and "CDR" are used interchangeably.

[0302] The term "constant region" or "constant domain" refers to the carboxyl-terminal portions of the light and heavy chains that are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as interacting with Fc receptors. The term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0303] The term "framework" or "FR" refers to variable domain residues that flank the CDRs. FR residues are present, for example, in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0304] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined to stretch from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0305] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); cytokine secretion, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays disclosed.

[0306] An "activating Fc receptor" is an Fc receptor that, upon binding by the Fc region of an antibody, stimulates the receptor-bearing cell to elicit signaling events that carry out effector functions. Exemplary activating Fc receptors include FcγRIIIα (CD16α), FcγRI (CD64), FcγRIIα (CD32), and FcαRI (CD89).

[0307] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature and which has not been manipulated, modified, and / or altered (e.g., isolated, purified, selected, included, or combined with other sequences, such as variable region sequences) by humans. Native sequence human IgG1 Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. For example, the native human IgG1 Fc region amino acid sequence is provided below: [ka] .

[0308] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, in the native-sequence Fc region or the Fc region of a parent polypeptide, compared to the native-sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region, as used herein, can have at least about 80% homology to the native-sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, e.g., at least about 95% homology thereto, e.g., to the variant.

[0309] A "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the change results from an alteration of the sequence containing said amino acid residue / position. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue / position, and / or deletion of said residue / position.

[0310] A "heterodimerization-promoting modification" is a manipulation of the peptide backbone or a post-translational modification of a peptide, e.g., an immunoglobulin heavy chain, that reduces or prevents the association of a polypeptide that forms a homodimer with an identical polypeptide. As used herein, heterodimerization-promoting modifications particularly include separate modifications made to each of two polypeptides desired to form a dimer, where the modifications are complementary to each other to promote the association of the two polypeptides. For example, a heterodimerization-promoting modification may alter the structure or charge of one or both of the polypeptides desired to form a dimer to sterically or electrostatically favor their association, respectively. Heterodimerization occurs between two non-identical polypeptides, e.g., two immunoglobulin heavy chains, where the additional immunoconjugate components (e.g., IL-2 polypeptides) fused to each of the heavy chains are not identical. In the immunoconjugates of the present disclosure, the heterodimerization-promoting modifications are in the heavy chains of the immunoglobulin molecule, specifically in the Fc domain. In some embodiments, the heterodimerization-promoting modifications comprise amino acid mutations, particularly amino acid substitutions, hi certain embodiments, the heterodimerization-promoting modifications comprise separate amino acid mutations, particularly amino acid substitutions, in each of the two immunoglobulin heavy chains.

[0311] The term "Fc domain" is used herein to define the C-terminal portion of an immunoglobulin composed of the Fc regions of both heavy chains of the immunoglobulin. Each heavy chain Fc region in an Fc domain is referred to herein as a subunit of the Fc domain. The two subunits of an Fc domain can be both native-sequence Fc regions, both variant Fc regions, or one native-sequence Fc region and one variant Fc region. In certain embodiments, the Fc domain contains a modification that promotes heterodimerization of two non-identical immunoglobulin heavy chains. The site of the most extensive protein-protein interaction between the two polypeptide chains of a human IgG Fc domain is in the CH3 domain of the Fc region. Thus, in one embodiment, the modification is in the CH3 domain of the Fc region. In a specific embodiment, the modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits, referred to as the "Fc-knob," and a hole modification in the other Fc subunit, referred to as the "Fc-hole." Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prat Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, so that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A cavity of the same or similar size as the protrusion and into which it fits is created by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine) at the interface of the second polypeptide. The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two Fc subunits, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two Fc subunits. In a further specific embodiment, the Fc subunit comprising the knob modification additionally comprises the amino acid substitution S354C, and the immunoglobulin heavy chain comprising the hole modification additionally comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two heavy chains, further stabilizing the dimer (Carter, J. Immunol Methods 248, 7-15 (2001)).

[0312] The term "variant," when used in reference to a peptide, polypeptide, or antibody, can refer to a peptide or polypeptide that contains one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5, etc.) amino acid sequence substitutions, deletions, and / or additions compared to the native or unmodified sequence. For example, an IL-2 variant can result from one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5, etc.) changes to the amino acid sequence of native IL-2. Also, by way of example, a variant of an anti-FAP antibody can result from one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5, etc.) changes to the amino acid sequence of a native or previously unmodified anti-FAP antibody. The variants may be naturally occurring, e.g., allelic or splice variants, or may be artificially constructed. Polypeptide variants may be prepared from corresponding nucleic acid molecules encoding the variants. In specific embodiments, the IL-2 variants or anti-FAP antibody variants retain at least the functional activity of the IL-2 or anti-FAP antibody, respectively. In specific embodiments, the anti-FAP antibody variants are bispecific antibodies that bind to both FAP and IL-2. In certain embodiments, the variants are encoded by single nucleotide polymorphism (SNP) variants in nucleic acid molecules encoding the VH or VL regions or subregions, e.g., one or more CDRs, of the IL-2 or anti-FAP antibody.

[0313] An "intact" antibody is one that contains not only the antigen-binding site but also the CL and at least the CH1, CH2, and CH3 heavy chain constant regions. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, the intact antibody has one or more effector functions.

[0314] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments; diabodies and di-diabodies (e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2003, Nat. Med. 9:129-34; WO93 / 11161; and U.S. Patent Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Patent Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Patent No. 7,612,181); single domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49); and multispecific antibodies formed from antibody fragments.

[0315] A "functional fragment," "binding fragment," or "antigen-binding fragment" of a therapeutic antibody refers to at least one biological function, some or all of which is not attributable to the intact antibody, where the function includes at least binding to a target antigen (e.g., an IL-2 binding fragment or a fragment that binds to IL-2).

[0316] As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one cytokine moiety and at least one antigen-binding moiety. In certain embodiments, an immunoconjugate comprises at least one cytokine moiety (e.g., IL-2) and at least two antigen-binding moieties (e.g., a masking moiety and an anchoring moiety described herein). Specifically, in certain embodiments, an immunoconjugate according to the present disclosure comprises one cytokine moiety and two antigen-binding moieties joined by one or more linker sequences. In certain embodiments, an immunoconjugate according to the present disclosure comprises one cytokine moiety and two antigen-binding moieties joined by an immunoglobulin Fc domain. In various embodiments of the present disclosure, the antigen-binding moiety can be joined to the cytokine moiety by various interactions and in various configurations described herein.

[0317] The terms "fusion," "fused," or other grammatical variations thereof, when used in reference to a peptide or polypeptide, or an antibody, refer to the conjugation of the peptide or polypeptide, or a fragment, variant, and / or derivative thereof, with a heterologous peptide or polypeptide.

[0318] An "affinity matured" antibody is one that has one or more alterations (e.g., amino acid sequence mutations, including alterations, additions, and / or deletions) in one or more HVRs thereof that result in improved affinity of the antibody for the antigen compared to a parent antibody that does not have such alterations. Affinity matured antibodies may have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For reviews, see Hudson and Souriau, 2003, Nature Medicine 9:129-34; Hoogenboom, 2005, Nature Biotechnol. 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51.

[0319] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate quickly, while high affinity antibodies generally bind antigen quickly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Specific illustrative embodiments include the following: In one embodiment, "K D " or "K D The "K value" may be measured by assays known in the art, for example, by binding assays. D K may be measured, for example, in an RIA performed using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). D or K D Values ​​may also be measured by using surface plasmon resonance assays by Biacore®, for example using a Biacore® TM-2000 or Biacore® TM-3000, or by biolayer interferometry, for example using an Octet® QK384 or Gator™ system. "On-rate" or "rate of association" or "association rate" or "k on" may also be determined using the same surface plasmon resonance or biolayer interferometry techniques described above, for example using a Biacore® TM-2000 or Biacore® TM-3000, or Octet® QK384, or Gator™ system.

[0320] The terms "inhibition" or "inhibiting" as used herein refer to partial (1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%, etc.) or complete (i.e., 100%) inhibition.

[0321] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native-sequence human FcR. Exemplary FcRs also bind IgG antibodies (e.g., gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains (see, e.g., Daeron, 1997, Annu. Rev. Immunol. 15:203-34). Various FcRs are known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9:457-92; Capel et al., 1994, Immunomethods 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med. 126:330-41). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (see, e.g., Guyer et al., 1976, J. Immunol. 117:587-93; and Kim et al., 1994, Eu. J. Immunol. 24:2429-34). Antibody variants with improved or diminished binding to FcRs have been described (see, e.g., WO2000 / 42072; U.S. Patent Nos. 7,183,387; 7,332,581; and 7,335,742; Shields et al. 2001, J. Biol. Chem. 9(2):6591-604).

[0322] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an antibody or cytokine polypeptide described herein, for introducing a nucleic acid sequence into a host cell. Vectors that can be used include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can contain selectable sequences or markers operable for stable integration into a host cell chromosome. In addition, a vector can contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included provide, for example, resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply important nutrients not present in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are coexpressed (e.g., both antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acids can be operably linked to one common expression control sequence, or can be linked to different expression control sequences, for example, one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid molecule will be expressed in an amount sufficient to produce the desired product (e.g., the anti-FAP antibody described herein), and will further understand that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0323] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that has been substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source. An "isolated" nucleic acid molecule, e.g., a cDNA molecule, may also be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding the antibodies described herein are isolated or purified. The terms encompass nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or clonal DNA isolates, and chemically synthesized analogs or heterologous biologically synthesized analogs. A substantially pure molecule can include isolated forms of the molecule.

[0324] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. "Oligonucleotide," as used herein, generally refers simply to a single-stranded synthetic polynucleotide, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells that produce the antibodies of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Suitable host cells are disclosed below.

[0325] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of a nascent RNA transcript is referred to as the transcription direction; the region of the sequence on the DNA strand that is 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence"; and the region of the sequence on the DNA strand that is 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0326] The term "nucleic acid encoding," or its grammatical equivalents, when used in reference to a nucleic acid molecule, refers to a nucleic acid molecule in its native state or when engineered by methods well known to those of skill in the art that can be transcribed to produce mRNA, which can then be translated into polypeptides and / or fragments thereof. The antisense strand is the complement of such a nucleic acid molecule, and the coding sequence can be deduced therefrom.

[0327] The term "recombinant antibody" refers to an antibody prepared, expressed, created, or isolated by recombinant means. A recombinant antibody can be an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, an antibody isolated from an animal transgenic and / or transchromosomic for human immunoglobulin genes (e.g., mouse or cow) (see, e.g., Taylor et al., 1992, Nucl. Acids Res. 20:6287-95), or an antibody prepared, expressed, created, or isolated by any other means, including splicing of immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies can have variable and constant regions that include those derived from human germline immunoglobulin sequences (see, e.g., Kabat et al., supra). However, in certain embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0328] The term "composition" is intended to encompass a product containing specified ingredients (e.g., immunoconjugate molecules provided herein), optionally in specified amounts.

[0329] "Carrier," as used herein, includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat powdered milk, glycerol, propylene glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions containing formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990). The compositions containing the pharmaceutical compounds may also contain an antibody, for example, in isolated or purified form, together with a suitable amount of a carrier.

[0330] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly, in humans.

[0331] The term "excipient" refers to an inert substance commonly used as a diluent, vehicle, preservative, binder, or stabilizer, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkylsulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990), which is hereby incorporated by reference in its entirety.

[0332] The terms "subject" and "patient" can be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In specific embodiments, the subject is a human.

[0333] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., an immunoconjugate molecule described herein) that is external to the body to a patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art.

[0334] The term "effective amount," as used herein, refers to the amount of an antibody or pharmaceutical composition provided herein that is sufficient to bring about a desired outcome.

[0335] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0336] "Substantially all" refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0337] The phrases "substantially similar" or "substantially the same" mean that one of ordinary skill in the art would understand that the difference between two values ​​is not significant enough to justify the difference between the values ​​(e.g., K DA difference between two values ​​(e.g., one associated with an antibody of the present disclosure, and the other associated with a reference antibody) represents a sufficiently high degree of similarity between two numerical values ​​(e.g., one associated with an antibody of the present disclosure, and the other associated with a reference antibody) such that the difference is considered to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by the difference (value). For example, the difference between two values ​​can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5%, depending on the value for the reference antibody.

[0338] The phrases "substantially increased," "substantially reduced," or "substantially different," as used herein, refer to a sufficiently high degree of difference between two numerical values ​​(e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values ​​to be statistically significant within the context of the biological characteristic measured by the values. For example, the difference between the two values ​​can be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50%, depending on the value for the reference antibody. 5.3 Compositions and methods of making same

[0339] In one aspect of the present disclosure, a cytokine-containing immunoconjugate molecule is provided herein. In some embodiments, the immunoconjugate molecule is a fusion protein comprising a cytokine portion and a non-cytokine portion operably linked to each other. According to the present disclosure, the cytokine-containing immunoconjugate molecule is capable of delivering a cytokine and activating its cellular activity in a specific tissue or cellular location in a subject. For example, in some embodiments, cytokine activity is reduced or blocked when the immunoconjugate molecule is present in an environment lacking an activation signal for the cytokine. In some embodiments, cytokine activity is activated or enhanced when the immunoconjugate molecule is present in an environment containing or enriched with an activation signal for the cytokine. For example, in some embodiments, the immunoconjugate molecule is configured for tissue-specific distribution upon administration to a subject. In certain embodiments, the immunoconjugate molecule is enriched in a specific tissue or cellular environment that provides an activation signal for the cytokine, thereby specifically activating cytokine activity in such tissue or cellular environment.

[0340] In specific embodiments, the activation signal for a cytokine is the presence of a signal molecule in a target tissue or cellular environment where cytokine activity is activated. In some embodiments, the signal molecule is concentrated in the target tissue or cellular environment, but is present in lower amounts or concentrations in other non-target tissues or cellular environments. In some embodiments, the activation signal for a cytokine is the presence of a signal molecule in the target tissue or cellular environment at a concentration above a threshold. In some embodiments, the signal molecule can interact with the immunoconjugate molecule, thereby activating cytokine activity. In some embodiments, the signal molecule is a peptidic molecule.

[0341] In specific embodiments, the immunoconjugate molecule is configured for targeted delivery and activation of cytokine activity in cancerous tissues, such as tumors. In these embodiments, the signal molecule for activating the cytokine can be an antigen expressed or concentrated in cancerous tissues, e.g., in the tumor microenvironment. In specific embodiments, the activation signal for the cytokine is an antigen expressed on tumor cells. In other embodiments, the activation signal for the cytokine is an antigen expressed on cells in the tumor microenvironment, e.g., tumor stromal cells. In specific embodiments, the activation signal for the cytokine is a tumor-associated antigen.

[0342] In some embodiments, the non-cytokine portion of the immunoconjugate molecule comprises a masking moiety capable of binding to the cytokine moiety, whereby the masking moiety reduces or blocks cytokine activity. In some embodiments, the immunoconjugate molecule comprises an antibody or antigen-binding fragment thereof fused to a cytokine polypeptide, where the antibody or antigen-binding fragment thereof is capable of binding to the cytokine polypeptide and reduces or blocks cytokine activity.

[0343] In some embodiments, the intramolecular bond between the cytokine portion and the masking moiety of the immunoconjugate molecule is reversible, and thus, in some embodiments, the immunoconjugate molecule is capable of switching between an active and an inactive state of the cytokine due to the reversible binding and dissociation between the cytokine portion and the masking moiety.

[0344] In some embodiments, the masking moiety is a bispecific two-in-one antibody or binding fragment thereof, which can bind to the cytokine moiety and a second target antigen different from the cytokine. In specific embodiments, when the immunoconjugate molecule is in an environment where the second target antigen is not present, the masking moiety comprising the two-in-one antibody or antigen-binding fragment thereof binds to the cytokine moiety of the immunoconjugate molecule, thereby inhibiting cytokine activity. In specific embodiments, when the immunoconjugate molecule is in an environment where the second target antigen is present in an amount or concentration below a certain threshold, the masking moiety comprising the two-in-one antibody or antigen-binding fragment thereof binds to the cytokine moiety of the immunoconjugate molecule, thereby inhibiting cytokine activity. In various embodiments, the environment is a cellular environment or a tissue-specific environment. In certain embodiments, the environment is cancerous tissue or a tumor microenvironment. In certain embodiments, the second target antigen is an antigen expressed by cancer cells. In other embodiments, the second target antigen is an antigen expressed by cells in the tumor microenvironment, such as tumor stromal cells. In some embodiments, the second target antigen is a tumor-associated antigen.

[0345] In some embodiments, the masking moiety is a bispecific two-in-one antibody or binding fragment thereof, which can bind to the cytokine moiety and a second target antigen different from the cytokine. In specific embodiments, when the immunoconjugate molecule is in an environment where the second target antigen is present, the masking moiety comprising the two-in-one antibody or antigen-binding fragment thereof binds to the second antigen and dissociates from the cytokine moiety of the immunoconjugate molecule, thereby activating cytokine activity. In specific embodiments, when the immunoconjugate molecule is in an environment where the second target antigen is present in an amount or concentration above a certain threshold, the masking moiety comprising the two-in-one antibody or antigen-binding fragment thereof binds to the second antigen and dissociates from the cytokine moiety of the immunoconjugate molecule, thereby activating cytokine activity. In various embodiments, the environment is a cellular environment or a tissue-specific environment. In certain embodiments, the environment is a cancerous tissue or tumor microenvironment. In certain embodiments, the second target antigen is an antigen expressed by tumor cells. In other embodiments, the second target antigen is an antigen expressed by cells in the tumor microenvironment, e.g., tumor stromal cells. In some embodiments, the second target antigen is a tumor-associated antigen.

[0346] In specific embodiments, the immunoconjugate molecule of the present disclosure comprises a cytokine portion and a non-cytokine portion, wherein the cytokine portion comprises an interleukin-2 (IL-2) polypeptide, and the non-cytokine portion comprises a bispecific, two-in-one antibody capable of binding both the IL-2 polypeptide in the immunoconjugate molecule and a second target antigen that is not IL-2. In certain embodiments, the second target antigen is an antigen expressed by tumor cells. In other embodiments, the second target antigen is an antigen expressed by cells in the tumor microenvironment, e.g., tumor stromal cells. In some embodiments, the second target antigen is a tumor-associated antigen. In specific embodiments, the second target antigen is fibroblast activation protein (FAP). In more specific embodiments, the IL-2 polypeptide is a wild-type IL-2 polypeptide. In other embodiments, the IL-2 polypeptide is a mutant IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a human IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a simian IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a murine IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a mutant IL-2 polypeptide described herein. In a specific embodiment, the mutant IL-2 polypeptide is IL-2hex. Additional mutant IL-2 polypeptides that can be used in conjunction with the present disclosure can be found in U.S. Pat. Nos. 10,184,009 and 5,229,109, and International Patent Publication No. WO2012107417A1, the disclosures of each of which are incorporated herein by reference in their entireties.

[0347] In some embodiments, the non-cytokine portion of the immunoconjugate molecule comprises an anchoring moiety configured to tether the immunoconjugate molecule to a target location for delivery. Thus, in some embodiments, immunoconjugate molecules of the present disclosure having an anchoring moiety can achieve tissue-specific distribution after administration to a subject, for example, after systemic administration to a subject. In some embodiments, the anchoring portion of the immunoconjugate molecule can specifically bind to a target molecule present in the target location for delivery. In some embodiments, the anchoring portion of the immunoconjugate molecule comprises an antibody or antigen-binding fragment thereof that can bind to an antigen present in the target location for delivery, thereby tethering the immunoconjugate molecule to the target location for delivery.

[0348] In some embodiments, the target location of delivery is a cellular or tissue-specific environment, hi some embodiments, the target location of delivery also contains an activation signal for the cytokine of the immunoconjugate molecule, such that cytokine activity can be activated at the target location.

[0349] In certain embodiments, the target location for delivery is cancerous tissue or the tumor microenvironment. In some embodiments, the target location for delivery is a specific type of tissue or population of cells in a subject. In some embodiments, the anchoring portion of the immunoconjugate molecule comprises an antibody or antigen-binding fragment thereof that binds to an antigen expressed on cancer cells. Thus, in these embodiments, when administered to a subject with cancer, the immunoconjugate molecule can bind to a population of cancer cells in the subject. In some embodiments, the anchoring portion of the immunoconjugate molecule comprises an antibody or antigen-binding fragment thereof that binds to an antigen present in the tumor microenvironment, e.g., an antigen expressed on the surface of tumor cells or an antigen secreted by cells in the tumor microenvironment, e.g., tumor stromal cells. Thus, in these embodiments, when administered to a subject with a solid tumor, the immunoconjugate molecule can be concentrated in the tumor microenvironment in the subject.

[0350] In some embodiments, the immunoconjugate molecule of the present disclosure comprises a cytokine portion, a masking portion, and an anchoring portion operably connected to each other. In specific embodiments, the masking portion is a bispecific two-in-one antibody or antigen-binding fragment thereof capable of binding to both the cytokine portion and a second target antigen that is not a cytokine. In specific embodiments, the anchoring portion is an antibody or antigen-binding fragment thereof capable of binding to a third target antigen, for example, an antigen present at the target location of delivery for the immunoconjugate molecule. In some embodiments, the target location of delivery also contains the second target antigen in an amount sufficient to compete with the cytokine for binding to the masking portion, resulting in dissociation of the masking portion from the cytokine and activation of cytokine activity at the target location of delivery.

[0351] In some embodiments, when administered to a subject, the immunoconjugate molecule can achieve tissue-specific distribution and become concentrated in a target tissue or cellular environment in the subject that contains a sufficient amount of a third antigen. In specific embodiments, the target tissue or cellular environment also contains a second target antigen in an amount sufficient to compete with the cytokine for binding to the masking moiety, resulting in dissociation of the masking moiety from the cytokine and activation of cytokine activity in the target tissue or cellular environment.

[0352] In specific embodiments, the second and third target antigens recognized by the masking and anchoring moieties, respectively, of the immunoconjugate are the same antigen. In alternative embodiments, the second and third target antigens recognized by the masking and anchoring moieties, respectively, of the immunoconjugate are different antigens.

[0353] In specific embodiments, the cytokine portion comprises an interleukin-2 (IL-2) polypeptide, and the non-cytokine portion of the immunoconjugate molecule comprises a masking portion comprising a bispecific, two-in-one antibody capable of binding both the IL-2 polypeptide in the immunoconjugate molecule and a second target antigen that is not IL-2. In certain embodiments, the second target antigen is an antigen expressed by tumor cells. In other embodiments, the second target antigen is an antigen expressed by cells in the tumor microenvironment, e.g., tumor stromal cells. In some embodiments, the second target antigen is a tumor-associated antigen. In specific embodiments, the second target antigen is fibroblast activation protein (FAP). In specific embodiments, the non-cytokine portion of the immunoconjugate molecule further comprises an anchoring portion comprising an antibody or antigen-binding fragment capable of binding to a third target antigen that is not IL-2. In certain embodiments, the third target antigen is an antigen expressed by tumor cells. In some embodiments, the third target antigen is an antigen expressed by cells in the tumor microenvironment, e.g., tumor stromal cells. In some embodiments, the third target antigen is a tumor-associated antigen. In a specific embodiment, the third target antigen is fibroblast activation protein (FAP). In even more specific embodiments, the IL-2 polypeptide is a wild-type IL-2 polypeptide. In even more specific embodiments, the IL-2 polypeptide is a wild-type IL-2 polypeptide. In other embodiments, the IL-2 polypeptide is a mutant IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a human IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a monkey IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a mouse IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a mutant IL-2 polypeptide described herein. In a specific embodiment, the mutant IL-2 polypeptide is IL-2hex.Additional mutant IL-2 polypeptides that can be used in conjunction with the present disclosure can be found in U.S. Pat. Nos. 10,184,009 and 5,229,109, and International Patent Publication No. WO2012107417A1, the disclosures of each of which are incorporated herein by reference in their entireties.

[0354] In some embodiments, the immunoconjugate molecule comprises an anchoring moiety, a masking moiety, and a cytokine moiety operably linked to one another via a conjugate moiety. In some embodiments, the conjugate moiety comprises an immunoglobulin Fc domain composed of the Fc regions of both heavy chains of an immunoglobulin (each an Fc domain subunit). In some embodiments, the Fc domain is the Fc domain of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4).

[0355] In some embodiments, the two subunits of the Fc domain can both be native-sequence Fc regions. In some embodiments, the two subunits of the Fc domain can both be variant Fc regions. In some embodiments, the two subunits of the Fc domain can be one native-sequence Fc region and one variant Fc region. In certain embodiments, the Fc domain comprises a modification that promotes heterodimerization of two non-identical immunoglobulin heavy chains. The most extensive protein-protein interaction site between the two polypeptide chains of a human IgG Fc domain is in the CH3 domain of the Fc region. Thus, in one embodiment, the modification is in the CH3 domain of the Fc region. In a specific embodiment, the modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits and a hole modification in the other of the Fc subunits. Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prat Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, so that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A cavity of the same or similar size as the protrusion and into which it fits is created by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine) at the interface of the second polypeptide. The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two Fc subunits, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two Fc subunits. In a further specific embodiment, the Fc subunit comprising the knob modification additionally comprises the amino acid substitution S354C, and the immunoglobulin heavy chain comprising the hole modification additionally comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two heavy chains, further stabilizing the dimer (Carter, J. Immunol Methods 248, 7-15 (2001)).

[0356] In alternative embodiments, modifications that promote heterodimerization of two non-identical polypeptide chains include modifications that mediate electrostatic steering effects, such as those described in PCT Publication No. WO2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two polypeptide chains with charged amino acid residues, such that homodimer formation is electrostatically disfavored, while heterodimerization is electrostatically favored.

[0357] Without being bound by theory, it is believed that the Fc domain confers favorable pharmacokinetic properties to the immunoconjugate molecule, including a long serum half-life that contributes to good accumulation at the target site and a favorable tissue-to-blood distribution ratio. At the same time, the Fc domain may result in undesirable targeting of the immunoconjugate molecule to cells that express Fc receptors rather than to target antigen-bearing cells. In addition, coactivation of the Fc receptor signaling pathway can result in cytokine release, which, combined with the cytokine polypeptide in the immunoconjugate molecule and the long half-life of the immunoconjugate, can lead to excessive activation of cytokine receptors and severe side effects upon systemic administration. Consistent with this, conventional IgG-IL-2 immunoconjugates have been reported to be associated with infusion reactions (see, for example, King et al., J Clin Onal 22, 4463-4473 (2004)).

[0358] In certain embodiments, modifications to the Fc region of the antibody result in a reduction or elimination of the effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP, and / or CDC. In some embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP, and CDC. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. For example, substitutions into human IgG1 using IgG2 residues 233-236 and IgG4 residues 327, 330, and 331 have shown greatly reduced ADCC and CDC (see, e.g., Armour et al., 1999, Eur. J. Immunol. 29(8):2613-24; and Shields et al., 2001, J. Biol. Chem. 276(9):6591-604). Other Fc variants are provided elsewhere herein.

[0359] To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. The term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0360] Thus, in some embodiments, an Fc domain forming part of an immunoconjugate molecule according to the present disclosure is engineered to have reduced binding affinity to an Fc receptor. In one such embodiment, the Fc domain comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor. In one such embodiment, one or more such amino acid mutations are present in one of the two Fc subunits of the Fc domain. In another such embodiment, one or more such amino acid mutations are present in both of the two Fc subunits of the Fc domain. In various embodiments, such amino acid mutations reduce the binding affinity of the immunoconjugate to an Fc receptor by at least two-fold, at least five-fold, or at least ten-fold.

[0361] In some embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain comprising the immunoconjugate molecule to an Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, an immunoconjugate comprising an engineered immunoglobulin molecule exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to an immunoconjugate comprising a non-engineered immunoglobulin molecule.

[0362] In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an Fcγ receptor. More specifically, in some embodiments, the Fc receptor is an FcγRIIIα, FcγRI, or FcγRIIα receptor. In some embodiments, the binding of the Fc domain to each of these exemplary receptors is reduced. In some embodiments, the binding affinity of the Fc domain to a complement component is reduced. Specifically, in some embodiments, the binding affinity of the Fc domain to C1q is reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to said receptor, is achieved when an immunoconjugate comprising said Fc domain exhibits a binding affinity to FcRn that is greater than about 70% of that of an unengineered form of an immunoconjugate molecule comprising said unengineered form of Fc. The immunoglobulin, or an immunoconjugate comprising said immunoglobulin, may exhibit such affinity of greater than about 80%, or even greater than about 90%.

[0363] In some embodiments, the Fc domain forming part of the immunoconjugate molecule is not a native-sequence Fc domain and has at least one amino acid mutation in one of its Fc subunits. In some embodiments, the Fc domain forming part of the immunoconjugate molecule is not a native-sequence Fc domain and has at least one amino acid mutation in both of its Fc subunits. In some embodiments, the amino acid mutations in both Fc subunits of the Fc domain are the same mutations. In some embodiments, the amino acid mutations in the two Fc subunits of the Fc domain are different mutations. In some embodiments, the amino acid mutations are selected from amino acid substitutions, amino acid deletions, and amino acid insertions. In certain embodiments, one or both of the Fc subunits in the Fc domain of the immunoconjugate molecule contain one or more amino acid mutations at any one or more of amino acid positions 228, 233, 234, 235, 236, 265, 297, 329, 330, and 331 of the Fc subunit, where the numbering of residues in the Fc subunits is that of the EU index as in Kabat. In certain embodiments, such one or more amino acid substitutions include S228P. In certain embodiments, such one or more amino acid substitutions include E233P. In certain embodiments, such one or more amino acid substitutions include L234V or L234A. In certain embodiments, such one or more amino acid substitutions include L235A or L235E. In certain embodiments, such one or more amino acid deletions include ΔG236. In certain embodiments, such one or more amino acid substitutions include D265G. In certain embodiments, such one or more amino acid substitutions include N297A or N297D. In certain embodiments, such one or more amino acid substitutions include P329E, P329A, or P329G, particularly P329E. In certain embodiments, such one or more amino acid substitutions include A330S. In certain embodiments, such one or more amino acid substitutions include P331S.

[0364] In certain embodiments, the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations at positions E233P, L234V, L235A, ΔG236, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations E233P, L234V, L235A, ΔG236, P329S, A330S, and P331S.

[0365] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations at positions L234A, L235A, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234A, L235A, A330S, and P331S.

[0366] In certain embodiments, the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, P329, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, P329, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations E233P, L234V, L235A, ΔG236, P329E, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations E233P, L234V, L235A, ΔG236, P329E, A330S, and P331S.

[0367] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, P329, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, P329, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations at positions L234A, L235A, P329E, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234A, L235A, P329E, A330S, and P331S.

[0368] In certain embodiments, the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, and P329. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, and P329. In a specific embodiment, the Fc domain comprises amino acid mutations E233P, L234V, L235A, ΔG236, and P329E. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations E233P, L234V, L235A, ΔG236, and P329E.

[0369] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, and P329. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, and P329. In a specific embodiment, the Fc domain comprises amino acid mutations at positions L234A, L235A, and P329E. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234A, L235A, and P329E.

[0370] In certain embodiments, the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, D265, A330, and P331. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, D265, A330, and P331. In certain embodiments, the Fc domain comprises amino acid mutations E233P, L234V, L235A, ΔG236, D265G, A330S, and P331S. In specific embodiments, both of the two Fc subunits comprise amino acid mutations E233P, L234V, L235A, ΔG236, D265G, A330S, and P331S. In these embodiments, the Fc domain has reduced binding affinity to Fcγ receptors.

[0371] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, D265, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, D265, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, D265G, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations L234A, L235A, D265G, A330S, and P331S.

[0372] In certain embodiments, the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, D265, P329, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, D265, P329, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations E233P, L234V, L235A, ΔG236, D265G, P329E, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations E233P, L234V, L235A, ΔG236, D265G, P329E, A330S, and P331S.

[0373] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, D265, P329, A330, and P331. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, D265, P329, A330, and P331. In a specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, D265G, P329E, A330S, and P331S. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations L234A, L235A, D265G, P329E, A330S, and P331S.

[0374] In certain embodiments, the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, D265, and P329. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, D265, and P329. In a specific embodiment, the Fc domain comprises amino acid mutations E233P, L234V, L235A, ΔG236, D265G, and P329E. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations E233P, L234V, L235A, ΔG236, D265G, and P329E.

[0375] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, D265, and P329. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, D265, and P329. In a specific embodiment, the Fc domain comprises amino acid mutations at positions L234A, L235A, D265G, and P329E. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234A, L235A, D265G, and P329E.

[0376] In certain embodiments, the Fc domain comprises amino acid mutations at positions L234, L235, and P329. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, and P329. In a specific embodiment, the Fc domain comprises amino acid mutations at positions L234A, L235A, and P329G. In a specific embodiment, both of the two Fc subunits comprise amino acid mutations at positions L234A, L235A, and P329G.

[0377] According to the present disclosure, the immunoconjugate molecule comprises an anchoring moiety, a masking moiety, and a cytokine moiety operably linked to each other via a conjugate moiety. In a specific embodiment, the cytokine moiety comprises a cytokine polypeptide. In a specific embodiment, the masking moiety comprises a bispecific, two-in-one antibody or antigen-binding fragment capable of binding to a cytokine polypeptide and a second target antigen. In a specific embodiment, the anchoring moiety comprises an antibody or antigen-binding fragment thereof capable of binding to a third target antigen. In a specific embodiment, the conjugate moiety comprises an immunoglobulin Fc domain composed of two Fc regions of an immunoglobulin heavy chain (each Fc region is referred to as a subunit of the Fc domain or "Fc subunit"). In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of the two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the Fc subunits (Fc-knob) and a hole modification in the other of the Fc subunits (Fc-hole).

[0378] According to the present disclosure, in these embodiments, the cytokine portion, masking portion, and anchoring portion of the immunoconjugate molecule can be operably linked to each other via the conjugate portion in a variety of different configurations. In an exemplary embodiment, the cytokine portion comprises a cytokine polypeptide fused to the C-terminus of one Fc subunit. In an exemplary embodiment, the masking portion comprises an antibody or antigen-binding fragment thereof fused to the C-terminus of one Fc subunit. In an exemplary embodiment, the cytokine portion comprises a cytokine polypeptide fused to the C-terminus of one Fc domain subunit, and the masking portion comprises an antibody or antigen-binding fragment thereof fused to the C-terminus of the other Fc subunit. In some embodiments, the masking portion is fused to the C-terminus of the Fc subunit. In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of the two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other of the Fc subunits (Fc-hole subunit).

[0379] In an exemplary embodiment, the cytokine portion comprises a cytokine polypeptide fused to the C-terminus of one of the Fc subunits. In an exemplary embodiment, the masking portion comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of one of the Fc subunits. In an exemplary embodiment, the anchoring portion comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc subunits. In an exemplary embodiment, the cytokine portion comprises a cytokine polypeptide fused to the C-terminus of one of the Fc domain subunits, and the masking portion comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of the other Fc subunit. In an exemplary embodiment, the cytokine portion comprises a cytokine polypeptide fused to the C-terminus of one of the Fc domain subunits, the masking portion comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of the other Fc subunit, and the anchoring portion comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc domain subunits. In a specific embodiment, the anchoring moiety and the cytokine moiety are fused to the N-terminus and C-terminus, respectively, of the same Fc subunit. In a specific embodiment, the masking moiety and the cytokine moiety are fused to the N-terminus and C-terminus, respectively, of the same Fc subunit. In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other of the Fc subunits (Fc-hole subunit).

[0380] In an exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc subunits. In an exemplary embodiment, the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety. In an exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc subunits, and the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety. In an exemplary embodiment, the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc subunits. In an exemplary embodiment, the cytokine moiety comprises a cytokine polypeptide fused to the anchoring moiety. In an exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc subunits, and the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of the other Fc subunit, and the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety. In one exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the N-terminus of one Fc subunit, the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of the other Fc subunit, and the cytokine moiety comprises a cytokine polypeptide fused to the anchoring moiety. In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of the two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other of the Fc subunits (Fc-hole subunit).

[0381] In an exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of one Fc subunit. In an exemplary embodiment, the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety. In an exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of one Fc subunit, and the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety. In an exemplary embodiment, the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of one Fc subunit. In an exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of one Fc subunit, and the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the N-terminus of the other Fc subunit, and the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety. In a specific embodiment, the masking moiety and the anchoring moiety bind to the same Fc subunit. In a specific embodiment, the masking moiety and the anchoring moiety bind to different Fc subunits. In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification that comprises a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other of the Fc subunits (Fc-hole subunit).

[0382] In one exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the C-terminus of one of the Fc subunits. In one exemplary embodiment, the cytokine moiety comprises a cytokine polypeptide fused to the C-terminus of one of the Fc subunits. In one exemplary embodiment, the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the masking moiety. In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of the two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other of the Fc subunits (Fc-hole subunit).

[0383] In one exemplary embodiment, the masking moiety comprises a bispecific two-in-one antibody or antigen-binding fragment thereof fused to the N-terminus of one of the Fc subunits. In one exemplary embodiment, the cytokine moiety comprises a cytokine polypeptide fused to the N-terminus of one of the Fc subunits. In one exemplary embodiment, the anchoring moiety comprises an antibody or antigen-binding fragment thereof fused to the masking moiety. In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of the two Fc subunits. In a specific embodiment, the modification is a knob-into-hole modification comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other of the Fc subunits (Fc-hole subunit).

[0384] According to the present disclosure, in any of the embodiments described herein, different portions of the immunoconjugate molecule may be connected by a peptide linker sequence. In some embodiments, the peptide linker has at least 5 amino acid residues. In some embodiments, the peptide linker has at least 7 amino acid residues. In some embodiments, the peptide linker has at least 10 amino acid residues. In some embodiments, the peptide linker has at least 15 amino acid residues. In some embodiments, the peptide linker has at least 20 amino acid residues.

[0385] According to the present disclosure, in any of the embodiments described herein, non-limiting examples of antibodies forming part of the immunoconjugate molecule can be synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, and anti-idiotypic (anti-Id) antibodies. In some embodiments, the antibodies forming part of the immunoconjugate molecule are monoclonal antibodies. In any of the embodiments described herein, the antigen-binding fragments forming part of the immunoconjugate molecule can be functional fragments of antibodies that retain some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments such as IL-2-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments (including, e.g., monospecific, bispecific, etc.), F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, minibodies, and single-domain antibodies (VHHs or nanobodies). In specific embodiments, the immunoconjugate molecule can have any of configurations 1-20 shown in Figure 5.

[0386] For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a Fab fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is an ScFv fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a single domain (VHH) antibody.

[0387] For example, in specific embodiments, the antibody in the anchoring portion of the immunoconjugate molecule is a Fab fragment. For example, in specific embodiments, the antibody in the anchoring portion of the immunoconjugate molecule is an ScFv fragment. For example, in specific embodiments, the antibody in the anchoring portion of the immunoconjugate molecule is a single domain (VHH) antibody.

[0388] For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a Fab fragment, and the antibody in the anchoring portion of the immunoconjugate molecule is also a Fab fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a Fab fragment, and the antibody in the anchoring portion of the immunoconjugate molecule is an ScFv fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a Fab fragment, and the antibody in the anchoring portion of the immunoconjugate molecule is a single domain (VHH) fragment.

[0389] For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is an ScFv fragment, and the antibody in the anchoring portion of the immunoconjugate molecule is a Fab fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is an ScFv fragment, and the antibody in the anchoring portion of the immunoconjugate molecule is also an ScFv fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is an ScFv fragment, and the antibody in the anchoring portion of the immunoconjugate molecule is a single domain (VHH) fragment.

[0390] For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a single domain (VHH) antibody, and the antibody in the anchoring portion of the immunoconjugate molecule is a Fab fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a single domain (VHH) antibody, and the antibody in the anchoring portion of the immunoconjugate molecule is an ScFv fragment. For example, in a specific embodiment, the bispecific two-in-one antibody in the masking portion of the immunoconjugate molecule is a single domain (VHH) antibody, and the antibody in the anchoring portion of the immunoconjugate molecule is also a single domain (VHH) fragment.

[0391] In a specific embodiment, the bispecific two-in-one antibody or antigen-binding fragment thereof forming part of the immunoconjugate molecule is capable of binding to both an IL-2 polypeptide and a fibrosis activating protein (FAP). In a specific embodiment, the bispecific two-in-one antibody comprises a VH region, a VL region, a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 of the amino acid sequences set forth in Tables 1-4. Thus, in some embodiments, the two-in-one antibody or functional fragment thereof provided herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from (a) antibody D001, (b) antibody D002, (c) antibody D029, (d) antibody D003, (e) antibody D047, (f) antibody D049, (g) any one of light chain variants D029LV1, D029LV2, D029LV3, D029LV4, and D029LV5, (h) any one of heavy chain variants D029HV1, D029HV2, D029HV3, D029HV4, D029HV5, and D029HV6, or (i) antibody B10, as shown in Tables 1-2. In specific embodiments, the two-in-one antibody or functional fragment thereof provided herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from antibody D029-HV1LV1, antibody D029-HV2LV3, antibody D029-HV2LV4, antibody D029-HV1LV5, antibody D029-HV3LV2, antibody D029-HV4LV2, or antibody D029-HV6LV2.In some embodiments, the two-in-one antibody or functional fragment thereof provided herein comprises a VH and VL region selected from (a) antibody D001, (b) antibody D002, (c) antibody D029, (d) antibody D003, (e) antibody D047, (f) antibody D049, (g) any one of light chain variants D029LV1, D029LV2, D029LV3, D029LV4, and D029LV5, (h) any one of heavy chain variants D029HV1, D029HV2, D029HV3, D029HV4, D029HV5, and D029HV6, or (i) antibody B10, as shown in Tables 3-4. In specific embodiments, the two-in-one antibody or functional fragment thereof provided herein comprises the VH and VL regions from antibody D029-HV1LV1, antibody D029-HV2LV3, antibody D029-HV2LV4, antibody D029-HV1LV5, antibody D029-HV3LV2, antibody D029-HV4LV2, or antibody D029-HV6LV2. The nomenclature "D029-HVxLVx" refers to an antibody comprising a combination of VH and VL domain sequences of the corresponding number shown in Tables 3-4. For example, "D029-HV2LV3" refers to an antibody comprising the VH domain sequence of D029HV2 and the VL domain sequence of D029LV3 shown in Tables 3-4. Table 1. Two-in-one VL CDR amino acid sequences [Table 1] Table 2. Two-in-one VH CDR amino acid sequences [Table 2] Table 3. Two-in-one VL domain amino acid sequences [Table 3] Table 4. Two-in-one VH domain amino acid sequences [Table 4]

[0392] In specific embodiments, the anchoring portion of the immunoconjugate molecule comprises an antibody or antigen-binding fragment thereof that binds to a fibrosis activating protein (FAP). In specific embodiments, the anti-FAP antibody comprises the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of the amino acid sequences depicted in Tables 5-8. Thus, in some embodiments, the anti-FAP antibody or functional fragment thereof provided herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from (a) antibody 872-5, (b) antibody 872-59, (c) 872-70, (d) 872-5V1, or (e) VHH6, as depicted in Tables 5-6. In some embodiments, the anti-FAP antibodies or functional fragments thereof provided herein comprise the VH and VL regions from (a) antibody 872-5, (b) antibody 872-59, (c) 872-70, (d) 872-5V1, or (e) VHH6, as shown in Tables 7-8. Table 5. Anti-FAP VL CDR amino acid sequences [Table 5] Table 6. Anti-FAP VH CDR amino acid sequences [Table 6] Table 7. Anti-FAP VL domain amino acid sequence [Table 7] Table 8. Anti-FAP VH domain amino acid sequence [Table 8]

[0393] In one particular aspect, provided herein are IL-2-containing immunoconjugate molecules that modulate IL-2 activity by reversibly binding to and dissociating from a region of IL-2 that is responsible for binding to a specific IL-2R subunit. In some embodiments, the IL-2 polypeptide in the immunoconjugate molecule further comprises one or more mutations that alter the binding activity of the IL-2 polypeptide to a specific IL-2R subunit.

[0394] In some embodiments, the immunoconjugate molecule comprises an IL-2 polypeptide conjugated to a masking moiety, wherein the masking moiety comprises a two-in-one antibody or antigen-binding fragment thereof capable of binding to the IL-2 polypeptide and a first target antigen; the masking moiety, upon binding to the IL-2 polypeptide, blocks binding of the IL-2 polypeptide to the IL-2 receptor alpha subunit (IL-2Rα); the masking moiety, upon binding to the first target antigen, dissociates from the IL-2 polypeptide, thereby releasing the IL-2 polypeptide for binding to IL-2Rα, and the IL-2 polypeptide comprises one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rβ. In some embodiments, the IL-2 polypeptide further comprises one or more mutations that alter binding of the IL-2 polypeptide to IL-2Rγ.

[0395] In some embodiments, the immunoconjugate molecule comprises an IL-2 polypeptide conjugated to a masking moiety, wherein the masking moiety comprises a two-in-one antibody or antigen-binding fragment thereof capable of binding to the IL-2 polypeptide and a first target antigen; the masking moiety, upon binding to the IL-2 polypeptide, blocks binding of the IL-2 polypeptide to the IL-2 receptor alpha subunit (IL-2Rβ); the masking moiety, upon binding to the first target antigen, dissociates from the IL-2 polypeptide, thereby releasing the IL-2 polypeptide for binding to IL-2Rβ, and the IL-2 polypeptide comprises one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rα. In some embodiments, the IL-2 polypeptide further comprises one or more mutations that alter binding of the IL-2 polypeptide to IL-2Rγ.

[0396] In some embodiments, the masking moiety blocks binding of the IL-2 polypeptide to the IL-2R alpha subunit. In some embodiments, the masking moiety binds to an epitope of IL-2 that includes one or more of residues P34, K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, N71, L72, Q74, Y107, and D109 of IL-2.

[0397] In some embodiments, the masking moiety blocks binding of an IL-2 polypeptide to the IL-2R α subunit. In a specific embodiment, the masking moiety binds to an epitope of IL-2 recognized by an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 101 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102. In some embodiments, the masking moiety competes for binding to IL-2 with an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 101 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102. In some embodiments, the masking moiety comprises (a) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDR1), VL CDR2, and VL CDR3 of antibody B10 shown in Table 1; and / or (b) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1), VH CDR2, and VH CDR3 of antibody B10 shown in Table 2. In some embodiments, the masking portion comprises (a) a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 103, 17, and 104, respectively, and (b) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 105, 106, and 38, respectively. In some embodiments, the masking portion comprises (a) a light chain variable region (VL) comprising the VL of antibody B10 shown in Table 3; and / or (b) a heavy chain variable region (VH) comprising the VH of antibody B10 shown in Table 4. In some embodiments, the masking portion comprises a VL comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, the masking portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, the masking portion comprises (a) a VL comprising the amino acid sequence of SEQ ID NO: 101; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 102.

[0398] In some embodiments, the masking moiety blocks the binding of an IL-2 polypeptide to IL-2Rβ. In some embodiments, the masking moiety binds to an epitope of IL-2 that includes one or more of residues L12, Q13, E15, H16, L19, D20, M23, R81, D84, D87, N88, V91, I92, and E95 of IL-2. In some embodiments, the masking moiety binds to an epitope of IL-2 that is recognized by the antibody 5UTZ. In some embodiments, the masking moiety competes with the antibody 5UTZ for binding to IL-2.

[0399] In some embodiments, the IL-2 polypeptide of the immunoconjugate molecule comprises one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rα. In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rα are selected from K35E, R38A, R38E, R38D, F42A, F42K, K43E, Y45A, E61R, E62A, L72G, or a combination thereof. In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rα comprise any one, two, three, four, five, six, seven, or eight mutations selected from K35E, R38A, R38E, R38D, F42A, F42K, K43E, Y45A, E61R, E62A, and L72G. For example, in some embodiments, the one or more mutations that weaken binding of an IL-2 polypeptide to IL-2Rα include F42A. In some embodiments, the one or more mutations that weaken binding of an IL-2 polypeptide to IL-2Rα include K35E and F42A. In some embodiments, the one or more mutations that weaken binding of an IL-2 polypeptide to IL-2Rα include F42A, Y45A, and L72G. In some embodiments, the one or more mutations that weaken binding of an IL-2 polypeptide to IL-2Rα include R38D, K43E, and E61R. In some embodiments, the one or more mutations that weaken binding of an IL-2 polypeptide to IL-2Rα include R38A, F42A, Y45A, and E62A. In some embodiments, binding of the IL-2 polypeptide to the IL-2R α subunit is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to wild-type IL-2.In some embodiments, binding of an IL-2 polypeptide to the IL-2R α subunit is reduced by about 0.5% to 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, or 95% to about 99% compared to wild-type IL-2.

[0400] In some embodiments, the IL-2 polypeptide of the immunoconjugate molecule comprises one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rβ. In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rβ are selected from H16E, H16R, H16A, D20T, D20G, D20A, N88D, N88S, N88R, V91G, V91A, V91R, and V91S, or a combination thereof. In some embodiments, the one or more mutations that weaken binding of the IL-2 polypeptide to IL-2Rβ comprise any one, two, three, or four mutations selected from H16E, H16R, H16A, D20T, D20G, D20A, N88D, N88S, N88R, V91G, V91A, V91R, and V91S. In some embodiments, binding of the IL-2 polypeptide to the IL-2Rβ subunit is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to wild-type IL-2. In some embodiments, binding of an IL-2 polypeptide to the IL-2R α subunit is reduced by about 0.5% to 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, or 95% to about 99% compared to wild-type IL-2.

[0401] In some embodiments, the IL-2 polypeptide further comprises one or more mutations that alter binding of the IL-2 polypeptide to the IL-2R gamma chain (IL-2Rγ). In some embodiments, the one or more mutations that alter binding of the IL-2 polypeptide to IL-2Rγ are selected from L18R, Q22E, Q74H, L80F, R81D, L85V, I92F, T123A, Q126X (where X=H, M, K, R, E, S, G, A, C, D, I, or T), I129V, S130A, S130R, or a combination thereof. In some embodiments, the one or more mutations that alter binding of an IL-2 polypeptide to IL-2Rγ comprise any one, two, three, four, five, six, seven, eight, nine, ten, or eleven mutations selected from L18R, Q22E, Q74H, L80F, R81D, L85V, I92F, T123A, Q126X (where X=H, M, K, R, E, S, G, A, C, D, I, or T), I129V, S130A, and S130R. For example, in some embodiments, the one or more mutations that alter binding of an IL-2 polypeptide to IL-2Rγ comprise Q126T, Q74H, L80F, R81D, L85V, and I92F. In some embodiments, the one or more mutations that alter binding of the IL-2 polypeptide to IL-2Rγ include L18R, Q22E, Q126T, and S130R. In some embodiments, binding of the IL-2 polypeptide to the IL-2Rγ subunit is enhanced or reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% compared to wild-type IL-2. In some embodiments, binding of an IL-2 polypeptide to the IL-2R α subunit is reduced by about 0.5% to 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, or 95% to about 99% compared to wild-type IL-2.

[0402] In some embodiments, the IL-2-containing immunoconjugate molecule described herein further comprises an anchoring moiety described herein. In some embodiments, the anchoring moiety comprises an antibody or antigen-binding fragment thereof that specifically binds to a second target antigen. In some embodiments, the masking moiety dissociates from the IL-2 polypeptide in the presence of a first target antigen expressed on the surface of a first cell.

[0403] In some embodiments, the second target antigen is expressed on the surface of the first cell or a second cell adjacent to the first cell. In some embodiments, the first target antigen and the second target antigen are the same or different. In some embodiments, the first target antigen and / or the second target antigen is a tumor-associated antigen. In some embodiments, the first target antigen and the second target antigen are each independently selected from FAP, Her2, Her3, CD19, CD20, BCMA, PSMA, CEA, cMET, EGFR, CA-125, MUC-1, EpCAM, or Trop-2. In some embodiments, the first target antigen is a FAP.

[0404] In some embodiments, the IL-2-containing immunoconjugate molecules described herein further comprise a conjugate moiety described herein. 5.3.1 Polyclonal antibodies

[0405] The antibody forming part of the immunoconjugate molecule of the present disclosure may include a polyclonal antibody. Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, optionally, an adjuvant. Typically, the immunizing agent and / or adjuvant are injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may comprise a polypeptide or a fusion protein thereof (e.g., an IL-2 polypeptide or a FAP polypeptide). It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in a mammal being immunized with the protein and one or more adjuvants, or to immunize the animal with such a protein. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Ribi, CpG, Poly 1C, Freund's complete adjuvant, and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Immunization protocols can be selected by those skilled in the art without undue experimentation. The mammal can then be bled and the serum can be assayed for antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus. Additionally or alternatively, lymphocytes can be obtained from the immunized animal for fusion and preparation of monoclonal antibodies from hybridomas, as described below. 5.3.2 Monoclonal antibodies

[0406] The antibodies forming part of the immunoconjugate molecules of the present disclosure may alternatively be monoclonal antibodies made using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0407] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[0408] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0409] Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable, high-level antibody production by selected antibody-producing cells, and are sensitive to a selective medium that selects against unfused parent cells. Exemplary myeloma cell lines are mouse myeloma lines, such as SP-2 and derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol. 133:3001-05; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987)).

[0410] The culture medium in which hybridoma cells grow is assayed for the production of monoclonal antibodies against the antigen. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as RIA or ELISA. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson et al., 1980, Anal. Biochem. 107:220-39.

[0411] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of the cells into mice.

[0412] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using Protein A or Protein G-Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0413] DNA encoding a monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells that do not otherwise produce antibody protein, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., 1993, Curr. Opinion in Immunol. 5:256-62 and Pluckthun, 1992, Immunol. Revs. 130:151-88.

[0414] In some embodiments, an antibody that binds an epitope (1) comprises the amino acid sequence of a VH domain and / or the amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH and / or VL domains described herein under stringent conditions (e.g., hybridization to filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 0.2% SDS at about 68° C.), or other stringent hybridization conditions known to one of skill in the art. See, for example, Current Protocols in Molecular Biology Vol. I, 6.3.1-6.3.6 and 2.10.3 (Ausubel et al. eds., 1989).

[0415] In some embodiments, an antibody that binds a FAP epitope comprises the amino acid sequence of a VH CDR or a VL CDR encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH CDRs and / or VL CDRs set forth in Tables 5-6 under stringent conditions (e.g., hybridization to filter-bound DNA in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 0.2% SDS at about 68° C.), or under other stringent hybridization conditions known to those of skill in the art (see, e.g., Ausubel et al., supra).

[0416] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using, for example, the techniques described in Antibody Phage Display: Methods and Protocols (O'Brien and Aitken eds., 2002). In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0417] The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which the VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0418] VH and VL gene repertoires can be cloned separately by PCR and randomly recombined in phage libraries, as described in Winter et al., supra, which can then be screened for antigen-binding clones. Libraries derived from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self antigens and also self antigens without any immunization, as described in Griffiths et al., 1993, EMBO J 12:725-34. Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and achieve rearrangement in vitro, as described, for example, by Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-88.

[0419] Screening of the library can be achieved by various techniques known in the art. For example, antigens (e.g., IL-2 polypeptides, fragments, or epitopes) can be used to coat the wells of an adsorption plate, expressed in host cells attached to an adsorption plate, or used in cell sorting, conjugated to biotin for capture by streptavidin-coated beads, or any other method for panning a display library. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by the use of extended washing and monovalent phage display as described in Bass et al., 1990, Proteins 8:309-14 and WO92 / 09690, and by the use of low antigen coating density as described in Marks et al., 1992, Biotechnol. 10:779-83.

[0420] Antibodies that form part of the immunoconjugate molecules described herein can be obtained by designing a suitable antigen screening procedure to select for a phage clone of interest, followed by constructing a full-length antibody clone from the phage clone of interest and suitable constant region (e.g., Fc) sequences as described in Kabat et al., supra, using the VH and / or VL sequences (e.g., Fv sequences), or various CDR sequences from the VH and VL sequences.

[0421] In another embodiment, the antibody forming part of the immunoconjugate molecule is generated by the method described in Bowers et al., 2011, Proc Natl Acad Sci USA. 108:20455-60, for example, using the SHM-XHL™ platform (AnaptysBio, San Diego, CA). Briefly, in this approach, a fully human IgG library is constructed in a mammalian cell line (e.g., HEK293) as the starting library. Mammalian cells displaying immunoglobulins that bind to the target peptide or epitope are selected (e.g., by FACS sorting), and then activation-induced cytidine deaminase (AID)-induced somatic hypermutation is reproduced in vitro to expand the diversity of the initially selected antibody pool. After several rounds of affinity maturation, high-affinity, high-specificity antibodies are generated by coupling mammalian cell surface display with in vitro somatic hypermutation. Additional methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Patent Application Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference. 5.3.2.1 Antibody fragments

[0422] The present disclosure provides antibodies and antibody fragments that form part of immunoconjugate molecules. In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of fragments allows for rapid clearance and can lead to improved access to cells, tissues, or organs. For a review of certain antibody fragments, see Hudson et al., 2003, Nature Med. 9:129-34.

[0423] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing salvage receptor-binding epitope residues are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv) (see, e.g., WO93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fvs and scFvs have intact binding sites that lack constant regions; therefore, they may be suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins may be constructed to fuse an effector protein to either the amino or carboxy terminus of the scFv (see, e.g., Borrebaeck, ed., supra). An antibody fragment may also be a "linear antibody," e.g., as described in the above-cited references. Such linear antibodies may be monospecific or multispecific, such as bispecific.

[0424] The binding structure derived from smaller antibodies is a separate variable domain (V domain), also called a single variable domain antibody (sdAb). Certain species of organisms, such as camelids and cartilaginous fish, possess high-affinity single V-like domains attached to an Fc-equivalent domain structure as part of their immune systems (Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49). V-like domains (called VhH in camelids and V-NAR in sharks) typically display long surface loops that allow penetration of the target antigen cavity. They also stabilize the isolated VH domain by masking hydrophobic surface patches.

[0425] These VhH and V-NAR domains have been used to engineer sdAbs. Human V-domain variants have been designed using selection from phage libraries and other approaches that result in stable, high-binding VL- and VH-derived domains.

[0426] Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain an antigen-binding site that binds to an epitope (e.g., an IL-2 epitope or a FAP epitope). The immunoglobulin molecules provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0427] Antibody variants and derivatives include antibody functional fragments that retain the ability to bind to an epitope (eg, an IL-2 epitope or a FAP epitope). Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and comprising a portion of a light chain and a heavy chain cross-linked by a disulfide bond); Fab' (e.g., an antibody fragment containing a single antigen-binding domain comprising a Fab and an additional portion of a heavy chain through the hinge region); F(ab')2 (e.g., two Fab' molecules joined by an interchain disulfide bond in the hinge region of the heavy chain; the Fab' molecules may be directed against the same or different epitopes); bispecific Fab (e.g., a Fab molecule having two antigen-binding domains, each of which may be directed against a different epitope); a single chain containing a variable region, also known as scFv (e.g., the variable antigen-binding determining regions of a single light chain and heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv, or dsFv (e.g., the variable antigen-binding determining regions of a single light chain and heavy chain of an antibody linked together by a disulfide bond). camelized VH (e.g., the variable antigen-binding determining region of a single heavy chain of an antibody in which some amino acids in the VH interface are those found in the heavy chain of a naturally occurring camelid antibody); bispecific scFvs (e.g., scFv or dsFv molecules having two antigen-binding domains, each of which may be directed against a different epitope); diabodies (e.g., dimeric scFvs formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of a second scFv; the two antigen-binding regions of a diabody may be directed against the same or different epitopes); and triabodies (e.g., trimeric scFvs formed in a manner similar to diabodies, but in which three antigen-binding domains are created in a single complex; the three antigen-binding domains may be directed against the same or different epitopes). 5.3.2.2 Humanized Antibodies

[0428] In some embodiments, the antibodies forming part of the immunoconjugate molecules provided herein can be humanized antibodies that bind human and / or cynomolgus antigens (such as human IL-2 or human FAP), etc. For example, a humanized antibody of the present disclosure can comprise one or more CDRs set forth in Tables 1-2 and 5-6. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed, for example, by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36.

[0429] In some cases, humanized antibodies are constructed by CDR grafting, which involves grafting the amino acid sequences of six CDRs of parent non-human antibodies (e.g., rodents) onto human antibody frameworks. For example, Padlan et al. determined that only about one-third of the residues in CDRs actually contact antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In the technique of SDR grafting, only SDR residues are grafted onto human antibody frameworks (see, for example, Kashmiri et al., 2005, Methods 36:25-34).

[0430] The selection of human variable domains, both light and heavy, used in creating a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308; and Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is based on the most abundant human subclass, V. L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0431] In an alternative framework based on CDR comparison, called superhumanization, the homology of FR is not important.The method involves comparing non-human sequences with functional human germline gene repertoires.Then, the gene that encodes the same or closely related standard structure as the mouse sequence is selected.Next, among the genes that share the standard structure with non-human antibodies, the one with the highest homology in CDR is selected as FR donor.Finally, non-human CDR is grafted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0432] Furthermore, it is generally desirable that antibodies be humanized while retaining their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display predicted three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0433] Another method for antibody humanization is based on a measure of antibody humanization called human string content (HSC). This method compares mouse sequences with the repertoire of human germline genes, and scores differences as HSC. Then, the target sequence is humanized by maximizing its HSC, rather than using a measure of overall identity, to create multiple diverse humanized variants (Lazar et al., 2007, Mol. Immunol. 44:1986-98).

[0434] In addition to the methods described above, empirical methods can also be used to create and select humanized antibodies.These methods include methods based on creating a large library of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening techniques.Antibody variants can be isolated from phage, ribosome, and yeast display libraries and by bacterial colony screening (see, for example, Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0435] In the FR library approach, a collection of residue variants is introduced into a specific position of the FR, and then the library is screened to select the FR that best supports the grafted CDR. The substituted residues may include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol. 224:487-99), or may be from a more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272:10678-84).

[0436] In FR shuffling, instead of generating a combinatorial library of selected residue variants, entire FRs are combined with non-human CDRs (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The library may be screened for binding in a two-step process, first humanizing the VL, followed by humanizing the VH. Alternatively, a one-step FR shuffling process may be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0437] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and the sequential replacement of non-human fragments with a library of human FRs and evaluation of binding. It begins with the CDR3 regions of the non-human VH and VL chains and progressively replaces other regions of the non-human antibody with human FRs, including CDR1 and CDR2 of both VH and VL. This methodology typically results in the retention of epitopes and the identification of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0438] "Human engineering" methods involve modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by making specific changes to the antibody's amino acid sequence to generate modified antibodies that have reduced immunogenicity in humans but still retain the desired binding characteristics of the original non-human antibody. Generally, the techniques involve classifying amino acid residues in non-human (e.g., murine) antibodies as "low risk," "medium risk," or "high risk" residues. The classification is performed using an overall risk / reward calculation that evaluates the expected benefit (e.g., immunogenicity in humans) of making a particular substitution against the risk that the resulting substitution will affect antibody folding. Specific human amino acid residues to be substituted at given positions (e.g., low or medium risk) in a non-human (e.g., murine) antibody sequence can be selected by aligning the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or consensus human antibody sequence. Amino acid residues at low or medium risk positions in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence according to the alignment. Techniques for producing human engineered proteins are described in more detail in Studnicka et al., 1994, Protein Engineering 7:805-14; U.S. Patent Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication No. WO 93 / 11794. 5.3.2.3 Human antibodies

[0439] Human antibodies can be constructed by combining Fv clone variable domain sequences selected from a human-derived phage display library with known human constant domain sequences. Alternatively, the human monoclonal antibodies of the present disclosure can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, by Kozbor, 1984, J. Immunol. 133:3001-05; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al., 1991, J. Immunol. 147:86-95.

[0440] It is also possible to generate transgenic animals (e.g., mice) that can produce a full repertoire of human antibodies upon immunization without the production of endogenous immunoglobulins.Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, for example, Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; U.S. Patent Nos. 6,075,181 and 6,150,584; and Lonberg et al., 2005, Nature Biotechnol. 23:1117-25).

[0441] Alternatively, human antibodies may be prepared through immortalization of human B lymphocytes that produce antibodies against a target antigen (e.g., such B lymphocytes may be harvested from an individual or immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95 and U.S. Pat. No. 5,750,373).

[0442] Gene shuffling can also be used to derive human antibodies from non-human, e.g., rodent, antibodies, where the human antibodies have similar affinities and specificities to the starting non-human antibody. According to this method, also known as "epitope imprinting" or "guided selection," either the heavy or light chain variable region of a non-human antibody fragment obtained by the phage display technique described herein is replaced with a repertoire of human V domain genes, generating a population of non-human chain / human chain chimeric scFvs or Fabs. Antigen selection results in the isolation of non-human chain / human chain chimeric scFvs or Fabs, where the human chain restores the antigen-binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone (e.g., the epitope guides (imprints) the selection of the human chain partner). Repeating the process to replace the remaining non-human chains results in a human antibody (see, for example, PCT WO93 / 06213; and Osbourn et al., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides a completely human antibody without FR or CDR residues of non-human origin. Examples of directed selection for humanizing mouse antibodies against cell surface antigens include folate-binding protein present on ovarian cancer cells (see, for example, Figini et al., 1998, Cancer Res. 58:991-96) and CD147, which is highly expressed on hepatocellular carcinoma (see, for example, Bao et al., 2005, Cancer Biol. Ther. 4:1374-80).

[0443] A potential drawback of the guided selection approach is that shuffling one antibody chain while keeping the other constant can lead to epitope drift.CDR retention can be applied to maintain the epitope recognized by non-human antibodies (see, for example, Klimka et al., 2000, Br. J. Cancer. 83:252-60; and Beiboer et al., 2000, J. Mol. Biol. 296:833-49).In this method, non-human VH CDR3 is usually retained, since this CDR may be the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition.However, in some instances, the VH CDR3 and VL CDR3, as well as VH CDR2, VL CDR2, and VL CDR1 of non-human antibodies may be retained. 5.3.3 Antibody Variants

[0444] In some embodiments, amino acid sequence modifications of the antibodies forming part of the immunoconjugate molecules described herein are contemplated. For example, it may be desirable to improve the antibody's binding affinity and / or other biological properties, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, in addition to the specific antibodies provided herein, it is contemplated that antibody variants can be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art will recognize that amino acid changes can alter post-translational processes of antibodies, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

[0445] In some embodiments, the antibodies provided herein are chemically modified, for example, by covalent attachment of any type of molecule to the antibody. Antibody derivatives can include antibodies that have been chemically modified, for example, by increasing or decreasing glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, chemical cleavage, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. In addition, the antibodies can contain one or more non-standard amino acids.

[0446] Variations may be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide that result in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Amino ac...

Claims

1. (a) a cytokine moiety comprising a cytokine polypeptide having cytokine activity; (b) a masking moiety. An immunoconjugate molecule comprising: the masking moiety comprises a bispecific antibody or antigen-binding fragment thereof capable of binding to the cytokine polypeptide and a first target antigen; when bound to the cytokine polypeptide, the masking moiety reduces or inhibits the cytokine activity; Upon binding to the first target antigen, the masking moiety dissociates from the cytokine polypeptide, thereby activating the cytokine activity. Immunoconjugate molecules.

2. The masking moiety may be an intact antibody, Fab, Fab', F(ab') ２ 2. The immunoconjugate molecule of claim 1, comprising a VHH formed from an Fv, scFv, dsFv, diabody, triabody, tetrabody, or antibody fragment.

3. The immunoconjugate molecule of claim 1 , wherein the bispecific antibody is a two-in-one antibody.

4. The immunoconjugate molecule of claim 1 , wherein the first target antigen is a tumor-associated antigen.

5. The immunoconjugate molecule of claim 1 , wherein the first target antigen is fibrosis activating protein (FAP).

6. The immunoconjugate molecule of claim 1 , wherein the cytokine moiety comprises wild-type interleukin-2 (IL-2) or a mutant IL-2.

7. (c) an anchoring moiety comprising an antibody or antigen-binding fragment thereof that specifically binds to a second target antigen; The immunoconjugate molecule of claim 1, further comprising:

8. The immunoconjugate molecule of claim 7 , wherein the second target antigen is a tumor-associated antigen.

9. The immunoconjugate molecule of claim 7 , wherein the second target antigen is fibrosis activating protein (FAP).

10. The anchoring moiety is an intact antibody, Fab, Fab', F(ab') ２ 8. The immunoconjugate molecule of claim 7, comprising a VHH formed from an Fv, scFv, dsFv, diabody, triabody, tetrabody, or antibody fragment. (i) the bispecific antibody or antigen-binding fragment of the masking moiety is a Fab, scFv or VHH; or (ii) the antibody or antigen-binding fragment thereof of the anchoring moiety is a Fab, scFv or VHH; The immunoconjugate molecule of claim 7.

12. (d) a conjugate moiety that operably connects two or more of said cytokine moiety, said masking moiety, and said anchoring moiety. The immunoconjugate molecule of claim 7, further comprising:

13. The immunoconjugate molecule of claim 12 , wherein the conjugate moiety comprises an immunoglobulin Fc domain or a mutant thereof.

14. The immunoconjugate molecule of claim 13, wherein the immunoglobulin Fc domain comprises a first subunit and a second subunit that are two non-identical polypeptide chains; and the immunoglobulin Fc domain comprises a first modification that promotes heterodimerization of the two non-identical polypeptide chains.

15. The immunoconjugate molecule of claim 14, wherein the first modification is a knob-into-hole modification comprising a knob modification in the first subunit and a hole modification in the second subunit.

16. A composition comprising the immunoconjugate molecule of claim 1 and a pharma- ceutically acceptable carrier.

17. A pharmaceutical composition for activating a cytokine-mediated effect at a target site, comprising an immunoconjugate molecule comprising a cytokine and a masking moiety; the masking moiety comprises a two-in-one antibody or an antigen-binding fragment thereof that binds to the cytokine through an intramolecular interaction and inhibits the cytokine-mediated effect; the two-in-one antibody or antigen-binding fragment is capable of binding to a first target antigen at the target site; when the immunoconjugate molecule is at the target site, the two-in-one antibody or antigen-binding fragment binds to the first target antigen and dissociates from the cytokine; the cytokine-mediated effect is activated at the target site; Pharmaceutical compositions.

18. 18. The pharmaceutical composition of claim 17, wherein the immunoconjugate molecule further comprises an anchoring moiety; the anchoring moiety comprises an antibody or antigen-binding fragment thereof capable of binding to a second target antigen at the target site.

19. The pharmaceutical composition of claim 17 , wherein the immunoconjugate molecule is delivered to the target site, and delivering the immunoconjugate molecule comprises administering the immunoconjugate molecule to a subject.

20. 20. The pharmaceutical composition of claim 19, wherein the cytokine-mediated effect is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% less at a non-target site compared to the cytokine-mediated effect at the target site following administration of the immunoconjugate molecule to a subject.

21. 1. An immunoconjugate molecule comprising an IL-2 polypeptide conjugated to a masking moiety, the masking moiety comprises a two-in-one antibody or antigen-binding fragment thereof capable of binding to the IL-2 polypeptide and a first target antigen; said masking moiety, when bound to said IL-2 polypeptide, blocks binding of said IL-2 polypeptide to a first IL-2 receptor (IL-2R) subunit; said masking moiety, upon binding to said first target antigen, dissociates from said IL-2 polypeptide, thereby releasing said IL-2 polypeptide for binding to said first IL-2R subunit; Immunoconjugate molecules.

22. A pharmaceutical composition for activating a target cell expressing IL-2R, comprising the immunoconjugate molecule of claim 21, wherein upon binding of the IL-2 polypeptide to the IL-2R, the target cell is activated, Optionally, the target cell is an immune cell; Optionally, the target cells are effector T cells, memory T cells, regulatory T cells, or combinations thereof; Optionally, the target cells are CD4+ T cells, CD8+ T cells, helper T cells, cytotoxic T cells, SLECs (short-lived effector cells), MPECs (memory precursor effector cells), TEs (terminal effector cells), NKs (natural killer cells), NKTs (natural killer T cells), innate lymphoid cells (types I-III), or combinations thereof; Optionally, the target cells are natural Treg (nTreg) cells, induced Treg (iTreg) cells, or a combination thereof; Optionally, activation of said target cells is measured as: (a) increasing the proliferation or maturation of said target cells; Optionally, the proliferation or maturation of said target cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%; or (b) increasing the survival time of said target cells; Optionally, the survival time of said target cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%. Pharmaceutical compositions.

23. 22. A pharmaceutical composition for increasing secretion of a pro-inflammatory cytokine by a population of T cells, comprising the immunoconjugate molecule of claim 21, wherein the IL-2 polypeptide, upon binding, activates the T cells; Optionally, the cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof; Optionally, production of said cytokine is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000%. Pharmaceutical compositions.

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