SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 7, 2017, is named 39723-709_601_SL.txt and is 60,062 bytes in size.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term disclosed herein and a term in an incorporated reference, the term herein controls.

BACKGROUND

Antibodies are a class of agents known as “biologicals.” The source of antibodies can be a polyclonal supply, such as human or horse serum, or derived from a monoclonal source (single cell clone). With the technologic capability to control and select for specific antigen binding, monoclonal antibodies have yielded dramatic therapeutic benefits. However, the difficulty of generating specific antibodies for certain targets has limited the successes, and the potential for therapeutic agents remains largely untapped.

One impediment to the development of monoclonal antibodies for human therapy is the need to “humanize” such antibodies, which are generally made in mice, rats and rabbits. If human patients are administered such antibodies without humanized constant regions, they can suffer from “serum sickness,” meaning that an endogenous immune response is mounted by the recipient against the non-human antibody sequences. Humanizing monoclonal antibodies produced in research animals can avoid this problem. However, the cost in time and expense for humanization of antibodies can be considerable.

Nucleolin is expressed on the cell surface of chronic lymphocytic leukemia (CLL) cells, acute myeloid leukemia (AML) cells, some forms of breast carcinoma, as well as other tumors. As such, nucleolin constitutes a promising tumor antigen for targeting of therapeutics, including antibodies.

BRIEF SUMMARY

In some of many aspects, the present disclosure is directed to specific antibodies that immunologically recognize, bind to, and/or inactivate nucleolin. Also provided herein are light and heavy chain sequences of anti-nucleolin antibodies that may either be used directly to prepare anti-nucleolin antibodies (e.g., human and/or monoclonal), or can be used to provide specific sequence elements (e.g., CDR sequences) that may be incorporated into different, desired antibody backgrounds. Antibodies that comprise the sequences disclosed herein can be used in a wide range of therapies directed to disabling nucleolin.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain CDR1 having at least 60% sequence identity to amino acid sequence SEQ ID NO:42; a heavy chain CDR2 having at least 60% sequence identity to amino acid sequence YIS; a heavy chain CDR3 having at least 60% sequence identity to amino acid sequence DM; a light chain CDR1 having at least 60% sequence identity to amino acid sequence SEQ ID NO:65; a light chain CDR2 having at least 60% sequence identity to amino acid sequence SEQ ID NO:54; and a light chain CDR3 having at least 60% sequence identity to amino acid sequence SEQ ID NO:66. In some embodiments, the at least 60% sequence identity is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain CDR1 has an amino acid sequence that comprises SEQ ID NO:42; a heavy chain CDR2 has an amino acid sequence that comprises YIS; a heavy chain CDR3 has an amino acid sequence that comprises DM; a light chain CDR1 has an amino acid sequence that comprises SEQ ID NO:65; a light chain CDR2 has an amino acid sequence that comprises SEQ ID NO:54; and a light chain CDR3 has an amino acid sequence that comprises SEQ ID NO:66.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain CDR1 having at least 60% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS. 24 to 26; a heavy chain CDR2 having at least 60% sequence identity to an amino acid sequence selected from the group consisting of YIS and SEQ ID NOS. 30 to 32; a heavy chain CDR3 having at least 60% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS. 37 to 39; a light chain CDR1 having at least 60% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS. 46 to 48; a light chain CDR2 having at least 60% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS. 52 to 54; and a light chain CDR3 having at least 60% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS. 58 to 61. In some embodiments, the at least 60% sequence identity is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:24; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:30; the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:37; the light chain CDR1 has the amino acid sequence of SEQ ID NO:46; the light chain CDR2 has the amino acid sequence of SEQ ID NO:52; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:58.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:25; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:31; the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:38; the light chain CDR1 has the amino acid sequence of SEQ ID NO:47; the light chain CDR2 has the amino acid sequence of SEQ ID NO:53; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:59.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:26; the heavy chain CDR2 has the amino acid sequence of YIS; the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:39; the light chain CDR1 has the amino acid sequence of SEQ ID NO:48; the light chain CDR2 has the amino acid sequence of SEQ ID NO:54; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:60.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:24; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:32; the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:37; the light chain CDR1 has the amino acid sequence of SEQ ID NO:46; the light chain CDR2 has the amino acid sequence of SEQ ID NO:52; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:61.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain variable region (VH) that has at least 60% sequence identity to the amino acid sequence of SEQ ID NO:3; and a light chain variable region (VL) that has at least 60% sequence identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the at least 60% sequence identity is at least: 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a VH that has an amino acid sequence of SEQ ID NO:3; and a VL has the amino acid sequence of SEQ ID NO:12.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain that has at least 60% sequence identity to the amino acid sequence of SEQ ID NO:2; and a light chain that has at least 60% identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, the amino acid sequence of SEQ ID NO:2 is encoded by the nucleotide sequence of SEQ ID NO:1. In some embodiments, the amino acid sequence of SEQ ID NO:11 is encoded by the nucleotide sequence of SEQ ID NO:10. In some embodiments, the at least 60% sequence identity is at least: 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain that has at least 60% sequence identity to the amino acid sequence of SEQ ID NO:14; and a light chain that has at least 60% identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, the at least 60% sequence identity is at least: 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof that comprises a heavy chain with the amino acid sequence of SEQ ID NO:2; and a light chain with the amino acid sequence of SEQ ID NO:11. In some cases, provided herein is an isolated antibody or fragment thereof that comprises a heavy chain with the amino acid sequence of SEQ ID NO:14; and a light chain with the amino acid sequence of SEQ ID NO:11.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain CDR1 having at least 60% sequence identity to an amino acid sequence of DYF; a heavy chain CDR2 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:74; a heavy chain CDR3 having at least 60% sequence identity to an amino acid sequence of AR or SEQ ID NO:77; a light chain CDR1 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:84; a light chain CDR2 having at least 60% sequence identity to an amino acid sequence of NVS; and a light chain CDR3 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:91. In some embodiments, the at least 60% sequence identity is at least: 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain CDR1 has an amino acid sequence that comprises DYF; a heavy chain CDR2 has an amino acid sequence that comprises SEQ ID NO:74; a heavy chain CDR3 has an amino acid sequence that comprises AR or SEQ ID NO:77; a light chain CDR1 has an amino acid sequence that comprises SEQ ID NO:84; a light chain CDR2 has an amino acid sequence that comprises NVS; and a light chain CDR3 has an amino acid sequence that comprises SEQ ID NO:91.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a heavy chain CDR1 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:69 or SEQ ID NO:70; a heavy chain CDR2 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:73 or SEQ ID NO:74; a heavy chain CDR3 having at least 60% sequence identity to an amino acid sequence of AR, SEQ ID NO:77, or SEQ ID NO:78; a light chain CDR1 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:83 or SEQ ID NO:84; a light chain CDR2 having at least 60% sequence identity to an amino acid sequence of NVS or SEQ ID NO:87; and a light chain CDR3 having at least 60% sequence identity to an amino acid sequence of SEQ ID NO:90 or SEQ ID NO:91. In some embodiments, the at least 60% sequence identity is at least: 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:69; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:73; the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:77; the light chain CDR1 has the amino acid sequence of SEQ ID NO:83; the light chain CDR2 has the amino acid sequence of SEQ ID NO:87; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:90.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:70; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:74; the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:78; the light chain CDR1 has the amino acid sequence of SEQ ID NO:84; the light chain CDR2 has the amino acid sequence of NVS; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:90.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:70; the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:74; the heavy chain CDR3 has the amino acid sequence of AR; the light chain CDR1 has the amino acid sequence of SEQ ID NO:84; the light chain CDR2 has the amino acid sequence of NVS; and the light chain CDR3 has the amino acid sequence of SEQ ID NO:91.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a VH that has at least 60% sequence identity to an amino acid sequence of SEQ ID NO:17; and a VL that has at least 60% sequence identity to an amino acid sequence of SEQ ID NO:19. In some embodiments, the amino acid sequence of SEQ ID NO:17 is encoded by the nucleotide sequence of SEQ ID NO:16. In some embodiments, the amino acid sequence of SEQ ID NO:19 is encoded by the nucleotide sequence of SEQ ID NO:18. In some embodiments, the at least 60% sequence identity is at least: 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, or 100% sequence identity.

In some cases, provided herein is an isolated antibody or fragment thereof, wherein the isolated antibody or fragment thereof binds to human nucleolin and comprises: a VH has an amino acid sequence of SEQ ID NO:17; and a VL that has an amino acid sequence of SEQ ID NO:19.

In some cases, provided herein is an isolated antibody or fragment thereof that comprises any combination of a heavy chain CDR, VH, or fragment thereof disclosed herein and a light chain CDR, VL, or fragment thereof disclosed herein.

In some cases, provided herein is an isolated anti-nucleolin antibody or fragment thereof that binds to amino acid sequence SEQ ID NO:21. In some cases, provided herein is an isolated anti-nucleolin antibody or fragment thereof that binds to an epitope within residues G300 to E466 of amino acid sequence SEQ ID NO:20. In some embodiments, the epitope comprises an amino acid selected from the group consisting of E453, R457, D455, K348, K427, G426, K403, Y402, and any combination thereof. In some embodiments, the isolated antibody or fragment thereof comprises a light chain CDR1 that binds to E453, R457, or a combination thereof. In some embodiments, the isolated antibody or fragment thereof comprises a light chain CDR2 that binds to D455. In some embodiments, the isolated antibody or fragment thereof comprises a light chain CDR3 that binds to K348. In some embodiments, the isolated antibody or fragment thereof comprises a heavy chain CDR1 that binds to K427. In some embodiments, the isolated antibody or fragment thereof comprises a heavy chain CDR2 that binds to K427, G426, or a combination thereof. In some embodiments, the isolated antibody or fragment thereof comprises a heavy chain CDR3 that binds to K403, Y402, or a combination thereof. In some embodiments, the nucleolin is cell-surface nucleolin. In some embodiments, the isolated antibody or fragment thereof is human or humanized. In some embodiments, the isolated antibody or fragment thereof is an IgG antibody. In some embodiments, the isolated antibody or fragment thereof is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the isolated antibody or fragment thereof is a fragment that is a Fab fragment, a Fab′ fragment, a F(ab′)2fragment, a Fv fragment, a diabody, a linear antibody, a single-chain antibody, or a multispecific antibody formed from an antibody fragment. In some embodiments, the isolated antibody or fragment thereof is a fragment that comprises an antigen binding region. In some embodiments, the isolated antibody or fragment thereof is nontotoxic to normal cells or tissues. In some embodiments, the isolated antibody or fragment thereof is cytotoxic, e.g., to a tumor or cancer cell. In some embodiments, the isolated antibody or fragment thereof is cytotoxic in presence of human serum. In some embodiments, the isolated antibody or fragment thereof kills at least 10% of a population of tumor or cancer cells, when incubated with the population of tumor or cancer cells for a period of time. In some embodiments, the isolated antibody or fragment thereof kills at least: 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; or about 100% of the population of tumor or cancer cells. In some embodiments, the incubation is in presence of human serum. In some embodiments, the period of time is about 48-96 hours. In some embodiments, the tumor or cancer cell comprises one, two, or more types of cancer cells selected from the group consisting of human lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer cells. In some embodiments, the tumor or cancer cell comprises one, two, or more types of cancer cells selected from the group consisting of A549, A375, MCF-7, Hep3B, HCT-116, NCI-H358, 786-0, DU-145, MDA-MB-231, MV4-11, U251, CG-EMT, MIA-PaCa2, and PANC-1 cells. In some embodiments, the population of tumor or cancer cells comprises breast cancer cells, e.g., MCF-7 or MDA-MB-231 cells. In some embodiments, the population of tumor or cancer cells comprises acute myeloid leukemia (AML) cells, e.g., MV4-11 cells. In some embodiments, the population of tumor or cancer cells comprises prostate cancer cells, e.g., DU-145 cells, or CG-EMT cells. In some embodiments, the population of tumor or cancer cells comprises lung cancer cells, e.g., A549 or NCI-H358 cells. In some embodiments, the population of tumor or cancer cells comprises skin malignant melanoma cells, e.g., A375 cells. In some embodiments, the population of tumor or cancer cells comprises hepatocellular carcinoma cells, e.g., Hep3B cells. In some embodiments, the population of tumor or cancer cells comprises colon cancer cells, e.g., HCT-116 cells. In some embodiments, the population of tumor or cancer cells comprises renal cancer cells, e.g., 786-0 cells. In some embodiments, the population of tumor or cancer cells comprises brain tumor cells, e.g., U251 cells. In some embodiments, the population of tumor or cancer cells comprises pancreas carcinoma cells, e.g., MIA-Paca2 or PANC-1 cells.

In some cases, provided herein is a recombinant cell that produces an isolated antibody or fragment thereof disclosed herein. In some cases, provided herein is an isolated nucleic acid encoding an isolated antibody or fragment thereof disclosed herein. In some cases, provided herein is a vector that comprises a nucleic acid disclosed herein. In some cases, provided herein is a host cell that comprises a nucleic acid disclosed herein or a vector disclosed herein. In some cases, provided herein is a method of producing an antibody or fragment thereof that comprises culturing a host cell disclosed herein so that the antibody or fragment thereof is produced.

In some cases, provided herein is a pharmaceutical composition that comprises an effective amount of an isolated antibody or fragment thereof disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the isolated antibody or fragment thereof is a monoclonal antibody. In some embodiments, the isolated antibody or fragment thereof is a polyclonal antibody.

In some cases, provided herein is a method of treating a cancer, comprising administering to a subject in need thereof a pharmaceutical composition that comprises an isolated antibody or fragment thereof disclosed herein. In some embodiments, the administering is injection. In some embodiments, the administering is intravenous or subcutaneous injection. In some embodiments, the administering occurs 1-3 times per week. In some embodiments, the method reduces a size of tumor in the subject by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the tumor is a solid tumor. In some embodiments, the pharmaceutical composition is dosed from 0.15 mg to 3 mg per kg of body weight of the subject, e.g., 0.5 mg to 2 mg per kg of body weight of the subject. In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the cancer comprises one, two, or more types of cancer selected from the group consisting of human lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer.

In some cases, provided herein is a method of killing cancer cells, comprising contacting with the cancer cells an isolated antibody or fragment thereof disclosed herein. In some cases, provided herein is a use of an isolated antibody or fragment thereof disclosed herein for treating cancer or killing cancer cells.

In some cases, provided herein is a use of an isolated antibody or fragment thereof disclosed herein in the manufacture of a medicament. In some embodiments, the medicament is for treatment of a cancer. In some embodiments, the medicament is for killing cancer cells.

In some cases, provided herein is a recombinant mammalian cell line, wherein the recombinant mammalian cell line comprises one or more cells that comprise a first nucleic acid sequence of SEQ ID NO:1 and a second nucleic acid sequence of SEQ ID NO:10. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are in a same construct. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are recombinantly or synthetically produced and cloned into an expression vector. In some embodiments, the expression vector is a pTT5 expression vector. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are transfected into the one or more cells.

In some cases, provided herein is a method of activating an immune system in a human subject, comprising administering to the subject an isolated antibody or fragment thereof disclosed herein. In some cases, provided herein is a method of treating cancer by inhibiting Transforming growth factor beta (TGFβ) in a human subject, comprising administering to the subject an isolated antibody or fragment thereof disclosed herein, whereby the TGFβ is inhibited and the cancer is treated. In some embodiments, the TGFβ is TGFβ1, TGFβ2, or TGFβ3.

In some cases, provided herein is a method of treating a cancer by preventing stabilization of an oncogenic mRNA in a human subject, comprising administering to the subject an isolated antibody or fragment thereof disclosed herein, whereby the oncogenic mRNA is destabilized and the cancer is treated. In some embodiments, the oncogenic mRNA is tumor protein p53 mRNA, B-cell lymphoma-extra large (Bcl-XL) mRNA, (B-cell lymphoma 2) Bcl-2 mRNA, gastrin mRNA, (growth arrest and DNA damage-inducible alpha) Gadd45α mRNA, matrix metallopeptidase 9 (MMP9) mRNA,Arabidopsis thalianakinesin (Atk1) mRNA, Cyclin 1 mRNA, interleukin-2 (IL-2) mRNA, prostaglandin H synthase-1 (Pghs-1) mRNA, or any combination thereof. In some cases, provided herein is a method of treating a cancer by reducing an expression level of an oncogenic protein in a human subject, comprising administering to the subject an isolated antibody or fragment thereof disclosed herein, whereby the expression level of the oncogenic protein is destabilized and the cancer is treated. In some embodiments, the oncogenic protein is tumor protein p53, Bcl-xL, Bcl-2, gastrin, Gadd45a, MMP9, Atk1, Cyclin 1, IL-2, Pghs-1, or any combination thereof. In some embodiments, the cancer comprises one, two, or more types of cancers selected from the group consisting of human lung cancer, skin cancer, breast cancer, liver cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, leukemia, brain cancer, and pancreas cancer.

In some cases, provided herein is an immunoconjugate, wherein the immunoconjugate comprises an antigen binding agent linked to a therapeutic agent, and wherein the antigen binding agent comprises an isolated antibody or fragment thereof disclosed herein. In some embodiments, the immunoconjugate is a fusion protein, and the therapeutic agent is a polypeptide. In some embodiments, the antigen binding agent is a bispecific antibody. In some embodiments, the antigen binding agent is a probody. In some embodiments, the probody comprises an antigen-binding region that is activated by a tumor cell. In some embodiments, the antigen-binding region comprises a peptide linked to the N-terminus of a light chain through a protease cleavable linker. In some embodiments, the antigen binding agent is linked covalently, noncovalently, or recombinantly to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the cytotoxic agent is doxorubicin, calicheamicin, auristatin, maytansinoid, brentuximab vedotin, tubulysins, duocarmycins, camptothecin, SN-38, pyrrolobenzodiazepine, methotrexate, α-amanitin, ansamitocin, or any combination thereof. In some embodiments, the therapeutic agent is an immune stimulating agent. In some embodiments, the therapeutic agent is interleukin-2 (IL-2), an immunostimulatory nucleic acid molecule, granulocyte macrophage colony-stimulating factor, resiquimod, Gardiquimod, phycocyanobilin, romiplostim, eltrombopag, or any combination thereof.

In some cases, provided herein is a pharmaceutical composition that comprises an immunoconjugate disclosed herein and a pharmaceutically acceptable carrier. In some cases, provided herein is a method of treating a cancer, comprising administering to a subject in need thereof a pharmaceutical composition that comprises an immunoconjugate disclosed herein. In some cases, provided herein is a use of an immunoconjugate disclosed herein for treating a cancer. In some cases, provided herein is a use of an immunoconjugate disclosed herein in the manufacture of a medicament for treatment of a cancer.

