Multifunctional molecules that bind to TCRs and uses thereof

Abstract

Provided herein are multifunctional polypeptide molecules comprising a T cell receptor variable beta binding portion and a cytokine, and methods of using same to treat a condition or disease in a subject.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 379,271, filed October 12, 2022, and U.S. Provisional Patent Application No. 63 / 381,231, filed October 27, 2022, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] Currently available molecules designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically target the CD3 epsilon (CD3e) subunit of the T cell receptor (TCR). However, this approach has limitations. Previous studies have shown that low doses of, for example, anti-CD3e monoclonal antibodies (mAbs) can cause T cell dysfunction and exert immunosuppressive effects. In addition, anti-CD3e mAbs bind to all T cells and thus activate large numbers of T cells. Such unphysiologically massive activation of T cells by these anti-CD3e mAbs can lead to the production of pro-inflammatory cytokines, such as IFN-gamma, IL-1-beta, IL-6, IL-10, and TNF-alpha, causing a "cytokine storm," also known as cytokine release syndrome (CRS), which is also associated with neurotoxicity (NT). Therefore, improved T cell receptor-binding molecules that redirect T cells for cancer immunotherapy are needed. Summary of the Invention [Means for solving the problem]

[0003]

[0003] Provided herein is a method for treating cancer in a human subject in need of cancer treatment, comprising the step of administering a multifunctional molecule to the human subject, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the multifunctional molecule is administered to the human subject at a first dose of about 0.001 mg / kg to about 10 mg / kg, thereby treating cancer in the human subject.

[0004]

[0004] Similarly, provided herein is a method for treating cancer in a human subject in need thereof, comprising the step of administering to the human subject a multifunctional molecule, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the administering step comprises administering multiple doses of the multifunctional molecule to the human subject.

[0005]

[0005] Similarly, provided herein is a method for treating cancer in a human subject in need thereof, comprising administering a first dose of a multifunctional molecule to the human subject, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, wherein the human subject is characterized by having a solid tumor, and where the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously been treated for symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or more, is not currently receiving treatment for CNS disease, and does not currently have leptomeningeal disease or spinal cord compression; and where the human subject has previously been treated with checkpoint inhibitor therapy (CPI), the human subject has remission of CPI immune-related toxicity to either grade ≦1 or baseline compared to before treatment with the CPI.

[0006]

[0006] Also provided herein is a method of treating cancer in a human subject in need thereof, comprising the step of administering to the human subject a first dose of a multifunctional molecule, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, wherein the human subject has no history of autoimmune disease; has not undergone major surgery or has had a traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject has no unhealed wounds from surgery or injury; has been treated with >10 mg / day of an immunosuppressant within 7 days prior to the first administration of the multifunctional molecule. have not been previously treated with cytotoxic chemotherapy, small molecule inhibitors, radiation therapy, or interventional radiology procedures within two weeks prior to the first administration of the multifunctional molecule; have not been previously treated with a monoclonal antibody, antibody-drug conjugate, or radioimmunoconjugate within six weeks prior to the first administration of the multifunctional molecule; do not have an inflammatory process that has not gone into remission within four weeks prior to the first administration of the multifunctional molecule; do not have clinically significant pulmonary dysfunction; or do not have an active viral, bacterial, or systemic fungal infection requiring parenteral treatment within seven days of the first administration of the multifunctional molecule.

[0007] In some embodiments, the first dose is a first dose of multiple doses. In some embodiments, the human subject is characterized by having a solid tumor.

[0008] In some embodiments, if the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously been treated for symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or more, and is not currently receiving treatment for CNS disease, and does not currently have leptomeningeal disease or spinal cord compression; if the human subject has previously been treated with checkpoint inhibitor therapy (CPI), the human subject has remission of CPI immune-related toxicity to either Grade ≦1 or baseline compared to before treatment with the CPI.

[0008]

[0009] In some embodiments, the solid tumor is selected from the group consisting of high mutational burden (TMB-H), microsatellite instability / DNA mismatch repair (MSI-H / dMMR), virus-associated tumors, metastatic triple-negative breast cancer (mTNBC), recurrent and refractory epithelial ovarian cancer, metastatic castration-resistant prostate cancer (mCRPC); K-Ras wild-type CRC; K-Ras mutant CRC, and primary stage IV or recurrent non-small cell lung cancer (NSCLC).

[0009]

[0010] In some embodiments, the virus-associated tumor comprises Merkel cell carcinoma, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

[0011] In some embodiments, the human subject is not concurrently being treated for a CNS disease, the subject does not have a leptomeningeal disease, or the subject does not have spinal cord compression.

[0010]

[0012] In some embodiments, the subject's CPI immune-related toxicity is grade ≦1 or baseline, and the subject is not experiencing CPI-associated endocrine abnormalities, or the subject is not experiencing CPI-associated grade 3-4 pneumonia, pericarditis / myocarditis, colitis and intestinal perforation, myositis, encephalitis, or peripheral neuropathy.

[0011]

[0013] In some embodiments, the human subject has no history of autoimmune disease other than vitiligo; psoriasis, atopic dermatitis, or other autoimmune skin conditions that do not require systemic treatment; Graves' disease that has been euthyroid for >4 weeks; hypothyroidism that is being controlled with thyroid hormone replacement therapy; hair loss; arthritis that is being managed without systemic treatment other than oral nonsteroidal anti-inflammatory drugs, and adrenal insufficiency that is well controlled with replacement therapy; has not undergone major surgery or has a traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject has no unhealed wounds from surgery or injury; has not undergone major surgery or has had a traumatic injury within 7 weeks prior to the first administration of the multifunctional molecule. Not previously treated with >10 mg / day of an inhibitor; Not previously treated with cytotoxic chemotherapy, small molecule inhibitors, radiation therapy, or interventional radiology procedures within 2 weeks prior to the first administration of the multifunctional molecule; Not previously treated with a monoclonal antibody, antibody-drug conjugate, or radioimmunoconjugate within 6 weeks prior to the first administration of the multifunctional molecule; Not have an inflammatory process that has not resolved within 4 weeks prior to the first administration of the multifunctional molecule; Not have clinically significant pulmonary dysfunction; Or Not have an active viral, bacterial, or systemic fungal infection requiring parenteral treatment within 7 days of the first administration of the multifunctional molecule.

[0012]

[0014] In some embodiments, the autoimmune disease does not include vitiligo; psoriasis, atopic dermatitis, or other autoimmune skin conditions that do not require systemic treatment; Graves' disease that has been euthyroid for >4 weeks; hypothyroidism that is being controlled with thyroid hormone replacement therapy; hair loss; arthritis that is being managed without systemic treatment other than oral nonsteroidal anti-inflammatory drugs, and adrenal insufficiency that is well controlled with replacement therapy.

[0013]

[0015] In some embodiments, the human subject is at least 18 years of age.

[0016] In some embodiments, the multifunctional molecule is administered to a human subject at a first dose of about 0.001 mg / kg to about 10 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.001 mg / kg to about 1 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.001 mg / kg to about 5 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.001 mg / kg to about 10 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.005 mg / kg to about 1 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.005 mg / kg to about 5 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.005 mg / kg to about 10 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.01 mg / kg to about 1 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.01 mg / kg to about 5 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.01 mg / kg to about 10 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.05 mg / kg to about 1 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.05 mg / kg to about 5 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.05 mg / kg to about 10 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.1 mg / kg to about 1 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.1 mg / kg to about 5 mg / kg. In some embodiments, the multifunctional molecule is administered at a first dose of about 0.1 mg / kg to about 10 mg / kg.

[0014]

[0017] In some embodiments, the multifunctional molecule is at 0.001 mg / kg, 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.20 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.30 mg / kg, 0.31 mg / kg, 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.40 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / g / kg, 0.15mg / kg, 0.16mg / kg, 0.17mg / kg, 0.18mg / kg, 0.19mg / kg, 0.2mg / kg, 0.21mg / kg, 0.22mg / kg, 0.23mg / kg, 0.24mg / kg, 0.25mg / kg, 0.26mg / kg, 0 .27mg / kg, 0.28mg / kg, 0.29mg / kg, 0.3mg / kg, 0.31mg / kg, 0.32mg / kg, 0.33mg / kg, 0.34mg / kg, 0.35mg / kg, 0.36mg / kg, 0.37mg / kg, 0.38mg / kg, 0.39mg / kg, 0.4mg / kg, 0.41mg / kg, 0.42mg / kg, 0.43mg / kg, 0.44mg / kg, 0.45mg / kg, 0.46mg / kg, 0.47mg / kg, 0.48mg / kg, 0.49mg / kg, 0.5mg / kg, 0.51mg / kg, 0.52 mg / kg, 0.53mg / kg, 0.54mg / kg, 0.55mg / kg, 0.56mg / kg, 0.57mg / kg, 0.58mg / kg, 0.59mg / kg, 0.6mg / kg, 0.61mg / kg, 0.62mg / kg, 0.63mg / kg, 0.64mg / kg, 0.65mg / kg, 0.66mg / kg, 0.67mg / kg, 0.68mg / kg, 0.69mg / kg, 0.7mg / kg, 0.71mg / kg, 0.72mg / kg, 0.73mg / kg, 0.74mg / kg, 0.75mg / kg, 0.76mg / kg, 0.77mg / kg, 0.78mg / kg, 0.79mg / kg, 0.8mg / kg, 0.81mg / kg, 0.82mg / kg, 0.83mg / kg, 0.84mg / kg, 0.85mg / kg, 0.86mg / kg, 0.87mg / kg, 0.88mg / kg, 0.89mg / kg, 0.9mg / kg、0.91mg / kg、0.92mg / kg、0.93mg / kg、0.94mg / kg、0.95mg / kg、0.96 mg / kg、0.97mg / kg、0.98mg / kg、0.99mg / kg、1mg / kg、1.5mg / kg、2mg / kg、2.5 mg / kg、3mg / kg、3.5mg / kg、4mg / kg、4.5mg / kg、5mg / kg、5.5mg / kg、6mg / kg、 6.5mg / kg、7mg / kg、7.5mg / kg、8mg / kg、8.5mg / kg、9mg / kg、9.5mg / kg、10mg / kg、10.5mg / kg、11mg / kg、11.5mg / kg、12mg / kg、12.5mg / kg、13mg / kg、13.5 mg / kg、14mg / kg、14.5mg / kg、15mg / kg、15.5mg / kg、16mg / kg、16.5mg / kg、17 mg / kg、17.5mg / kg、18mg / kg、18.5mg / kg、19mg / kg、19.5mg / kg、20mg / kg、20 .5mg / kg、21mg / kg、21.5mg / kg、22mg / kg、22.5mg / kg、23mg / kg、23.5mg / kg、 24mg / kg、24.5mg / kg、25mg / kg、25.5mg / kg、26mg / kg、26.5mg / kg、27mg / kg 、27.5mg / kg、28mg / kg、28.5mg / kg、29mg / kg、29.5mg / kg、30mg / kg、30.5mg / kg、31mg / kg、31.5mg / kg、32mg / kg、32.5mg / kg、33mg / kg、33.5mg / kg、34mg / kg、34.5mg / kg、35mg / kg、35.5mg / kg、36mg / kg、36.5mg / kg、37mg / kg、37.5 mg / kg、38mg / kg、38.5mg / kg、39mg / kg、39.5mg / kg、40mg / kg、40.5mg / kg、4 1mg / kg、41.5mg / kg、42mg / kg、42.5mg / kg、43mg / kg、43.5mg / kg、44mg / kg、4 4.5mg / kg、45mg / kg、45.5mg / kg、46mg / kg、46.5mg / kg、47mg / kg、47.5mg / kg 48mg / kg, 48.5mg / kg, 49mg / kg, 49.5mg / kg.

[0015]

[0018] In some embodiments, the administering step comprises administering multiple doses of the multifunctional molecule to the human subject.

[0019] In some embodiments, a subsequent dose of the multiple doses is a lower dose than the previous dose immediately preceding the subsequent dose and is administered after the previous dose has been shown to be intolerable. In some embodiments, a subsequent dose of the multiple doses is the same dose as the previous dose immediately preceding the subsequent dose and is administered after the previous dose has been shown to be tolerable. In some embodiments, a subsequent dose of the multiple doses is a higher dose than the previous dose immediately preceding the subsequent dose and is administered after the previous dose has been shown to be tolerable.

[0016]

[0020] In some embodiments, a subsequent dose of the multiple doses is the same dose as the previous dose immediately preceding the subsequent dose and is administered after the previous dose has been shown to be effective. In some embodiments, a subsequent dose of the multiple doses is lower than the previous dose immediately preceding the subsequent dose and is administered after the previous dose has been shown to be effective. In some embodiments, a subsequent dose of the multiple doses is higher than the previous dose immediately preceding the subsequent dose and is administered after the previous dose has been shown to be ineffective.

[0017]

[0021] In some embodiments, a subsequent dose of the multiple doses is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration in the preceding dose immediately preceding the subsequent dose. In some embodiments, a subsequent dose of the multiple doses is administered at least 1, 2, 3, or 4 weeks after administration in the preceding dose immediately preceding the subsequent dose. In some embodiments, a subsequent dose of the multiple doses is administered at least 1, 2, 3, 4, 5, 10, 11, or 12 months after administration in the preceding dose immediately preceding the subsequent dose.

[0018]

[0022] In some embodiments, the administration frequency of the multiple doses is maintained or reduced after the preceding dose immediately preceding the subsequent dose is shown to be effective. In some embodiments, the administration frequency of the administering step is increased after a dose of the multiple doses is shown to be ineffective. In some embodiments, the method comprises administering the multifunctional molecule to a human subject once a week.

[0019]

[0023] In some embodiments, the method comprises administering the multifunctional molecule to a human subject once a week for at least 1, 2, 3, or 4 weeks, or for at least 1, 2, 3, 4, 5, 10, 11, or 12 months, or for at least 1, 2, or 3 years. In some embodiments, the method comprises administering the multifunctional molecule to a human subject once every two weeks. In some embodiments, the method comprises administering the multifunctional molecule to a human subject once every two weeks for at least 1, 2, 3, or 4 weeks, or for at least 1, 2, 3, 4, 5, 10, 11, or 12 months, or for at least 1, 2, or 3 years. In some embodiments, the method comprises administering the multifunctional molecule to a human subject once every three weeks. In some embodiments, the method comprises administering the multifunctional molecule to a human subject once every three weeks for at least 1, 2, 3, or 4 weeks, or at least 1, 2, 3, 4, 5, 10, 11, or 12 months, or at least 1, 2, or 3 years. In some embodiments, the method comprises administering the multifunctional molecule to a human subject once every two weeks for 28 days, during which the multifunctional molecule is administered to the human subject on days 1 and 15.

[0020]

[0024] In some embodiments, the multifunctional molecule is administered by intravenous infusion. In some embodiments, the multifunctional molecule is administered subcutaneously, intratumorally, intranodally, intramuscularly, intradermally, or intraperitoneally.

[0021]

[0025] In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of about 25 minutes to about 240 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of about 105 minutes to about 120 minutes or about 125 minutes to about 145 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of about 150 minutes to about 200 minutes or about 160 minutes to about 190 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of about 25 minutes to about 35 minutes or about 55 minutes to about 65 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of about 35 minutes to about 50 minutes or about 85 minutes to about 95 minutes.

[0022]

[0026] In some embodiments, the method further comprises administering at least one additional therapeutic agent or treatment. In some embodiments, the at least one additional therapeutic agent or treatment is administered simultaneously with the dose of the multifunctional molecule. In some embodiments, the at least one additional therapeutic agent or treatment is administered before the dose of the multifunctional molecule. In some embodiments, the at least one additional therapeutic agent or treatment is administered after the dose of the multifunctional molecule.

[0023]

[0027] In some embodiments, the administering step comprises administering a pharmaceutical composition comprising the multifunctional molecule, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent that is saline. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent that is 0.9% saline.

[0024]

[0028] In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL. In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.2 mg / mL to about 15 mg / mL. In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 1.5 mg / mL. In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.2 mg / mL to about 1.5 mg / mL.

[0025]

[0029] In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at about 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.20 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.30 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.40 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, 0.50 mg / mL, 0.51 mg / mL, 0.52 mg / mL, 0.53 mg / mL, 0.54 mg / mL, 0.55 mg / mL, 0.56 mg / mL, 5mg / mL, 0.3mg / mL, 0.35mg / mL, 0.4mg / mL, 0.45mg / mL, 0.5mg / mL, 0.55mg / mL, 0.6mg / mL, 0.65mg / mL, 0.7mg / mL, 0.75mg / mL, 0.8mg / mL, 0.85mg / mL, 0.9mg / mL, 0.95mg / mL, 1mg / mL, 1.05mg / mL, 1.1mg / mL, 1.15mg / mL, 1.2mg / mL, 1.25mg / mL, 1.3mg / mL, 1.35mg / mL, 1.4 mg / mL, 1.45mg / mL, 1.5mg / mL, 1.55mg / mL, 1.6mg / mL, 1.65mg / mL, 1.7mg / mL, 1.75mg / mL, 1.8mg / mL, 1.85mg / mL, 1.9mg / mL, 1.95mg / mL, 2mg / mL, 2.05mg / mL, 2.1mg / mL, 2.15mg / mL, 2.2mg / mL, 2.25mg / mL, 2.3mg / mL, 2.35mg / mL, 2.4mg / mL, 2.45mg / mL, 2.5mg / mL, 3mg / mL, 3.5mg / mL, 4mg / mL, 4.5mg / mL, 5mg / mL, 5.5mg / mL, 6mg / mL, 6.5mg / mL, 7mg / mL, 7.5mg / mL, 8mg / mL, 8.5mg / mL, 9mg / mL, 9.5mg / mL, Present in concentrations of 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL, 13 mg / mL, 13.5 mg / mL, 14 mg / mL, 14.5 mg / mL, or 15 mg / mL.

[0026]

[0030] In some embodiments, the pharmaceutical composition comprises about 0.5 mL to about 500 mL of diluent.

[0031] Similarly, provided herein is a dose of a pharmaceutical composition comprising a multifunctional molecule, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and the dose is about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule.

[0027]

[0032] In some embodiments, the dose is about 0.001 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.001 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.005 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the multifunctional molecule is administered in a first dose of about 0.005 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.005 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.01 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.01 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.01 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.05 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.05 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.05 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.1 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.1 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dose is about 0.1 mg / kg to about 10 mg / kg of the multifunctional molecule.

[0028]

[0033] The dosage forms of the drugs are 0.001mg / kg, 0.002mg / kg, 0.003mg / kg, 0.004mg / kg, and 0. 005mg / kg、0.006mg / kg、0.007mg / kg、0.008mg / kg、0.009mg / kg、0.01mg / kg 、0.02mg / kg、0.03mg / kg、0.04mg / kg、0.05mg / kg、0.06mg / kg、0.07mg / kg、0 .08mg / kg、0.09mg / kg、0.1mg / kg、0.11mg / kg、0.12mg / kg、0.13mg / kg、0.14m g / kg、0.15mg / kg、0.16mg / kg、0.17mg / kg、0.18mg / kg、0.19mg / kg、0.2mg / kg g、0.21mg / kg、0.22mg / kg、0.23mg / kg、0.24mg / kg、0.25mg / kg、0.26mg / kg、0 .27mg / kg、0.28mg / kg、0.29mg / kg、0.3mg / kg、0.31mg / kg、0.32mg / kg、0.33 mg / kg、0.34mg / kg、0.35mg / kg、0.36mg / kg、0.37mg / kg、0.38mg / kg、0.39mg / kg、0.4mg / kg、0.41mg / kg、0.42mg / kg、0.43mg / kg、0.44mg / kg、0.45mg / kg、 0.46mg / kg、0.47mg / kg、0.48mg / kg、0.49mg / kg、0.5mg / kg、0.51mg / kg、0.52 mg / kg、0.53mg / kg、0.54mg / kg、0.55mg / kg、0.56mg / kg、0.57mg / kg、0.58mg / kg、0.59mg / kg、0.6mg / kg、0.61mg / kg、0.62mg / kg、0.63mg / kg、0.64mg / kg、 0.65mg / kg、0.66mg / kg、0.67mg / kg、0.68mg / kg、0.69mg / kg、0.7mg / kg、0.7 1mg / kg、0.72mg / kg、0.73mg / kg、0.74mg / kg、0.75mg / kg、0.76mg / kg、0.77mg / kg、0.78mg / kg、0.79mg / kg、0.8mg / kg、0.81mg / kg、0.82mg / kg、0.83mg / kg 、0.84mg / kg、0.85mg / kg、0.86mg / kg、0.87mg / kg、0.88mg / kg、0.89mg / kg、0.9mg / kg、0.91mg / kg、0.92mg / kg、0.93mg / kg、0.94mg / kg、0.95mg / kg、0.96 mg / kg、0.97mg / kg、0.98mg / kg、0.99mg / kg、1mg / kg、1.5mg / kg、2mg / kg、2. 5mg / kg、3mg / kg、3.5mg / kg、4mg / kg、4.5mg / kg、5mg / kg、5.5mg / kg、6mg / kg 、6.5mg / kg、7mg / kg、7.5mg / kg、8mg / kg、8.5mg / kg、9mg / kg、9.5mg / kg、10mg / kg、10.5mg / kg、11mg / kg、11.5mg / kg、12mg / kg、12.5mg / kg、13mg / kg、13. 5mg / kg、14mg / kg、14.5mg / kg、15mg / kg、15.5mg / kg、16mg / kg、16.5mg / kg、1 7mg / kg、17.5mg / kg、18mg / kg、18.5mg / kg、19mg / kg、19.5mg / kg、20mg / kg、 20.5mg / kg、21mg / kg、21.5mg / kg、22mg / kg、22.5mg / kg、23mg / kg、23.5mg / kg g、24mg / kg、24.5mg / kg、25mg / kg、25.5mg / kg、26mg / kg、26.5mg / kg、27mg / kg、27.5mg / kg、28mg / kg、28.5mg / kg、29mg / kg、29.5mg / kg、30mg / kg、30.5 mg / kg、31mg / kg、31.5mg / kg、32mg / kg、32.5mg / kg、33mg / kg、33.5mg / kg、3 4mg / kg、34.5mg / kg、35mg / kg、35.5mg / kg、36mg / kg、36.5mg / kg、37mg / kg、3 7.5mg / kg、38mg / kg、38.5mg / kg、39mg / kg、39.5mg / kg、40mg / kg、40.5mg / kg g、41mg / kg、41.5mg / kg、42mg / kg、42.5mg / kg、43mg / kg、43.5mg / kg、44mg / k g、44.5mg / kg、45mg / kg、45.5mg / kg、46mg / kg、46.5mg / kg、47mg / kg、47.5m g / kg, 48mg / kg, 48.5mg / kg, 49mg / kg, 49.5mg / kg.

[0029]

[0034] Also provided herein is a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and the pharmaceutically acceptable diluent is saline.

[0030]

[0035] In some embodiments, the pharmaceutically acceptable diluent is 0.9% saline.

[0036] In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL or about 0.2 mg / mL to about 1.5 mg / mL. In some embodiments, the total volume of the pharmaceutical composition is about 0.5 mL to about 500 mL. In some embodiments, the total volume of the pharmaceutical composition is about 5 mL to about 500 mL. In some embodiments, the total volume of the pharmaceutical composition is about 50 mL to about 500 mL. In some embodiments, the total volume of the pharmaceutical composition is about 0.5 mL to about 350 mL. In some embodiments, the total volume of the pharmaceutical composition is about 0.5 mL to about 250 mL. In some embodiments, the total volume of the pharmaceutical composition is about 0.5 mL to about 150 mL. In some embodiments, the total volume of the pharmaceutical composition is about 0.5 mL to about 50 mL.

[0031]

[0037] Also provided herein is a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL.

[0032]

[0038] Also provided herein is a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and the pharmaceutical composition comprises about 0.1 mg to about 500 mg of the multifunctional molecule.

[0033]

[0039] In some embodiments, the pharmaceutical composition comprises about 0.5 mg to about 200 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 0.5 mg to about 100 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 1 mg to about 200 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 1 mg to about 100 mg of the multifunctional molecule.

[0034]

[0040] Also provided herein is a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable excipient, wherein the multifunctional molecule comprises a TCRβV6 binding portion and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and the pharmaceutically acceptable excipient comprises one or more of L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

[0035]

[0041] In some embodiments, the pharmaceutical composition comprises about 0.1 mg to about 500 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 0.5 mg to about 200 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 0.5 mg to about 100 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 1 mg to about 200 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises about 1 mg to about 100 mg of the multifunctional molecule.

[0036]

[0042] In some embodiments, the pharmaceutical composition comprises about 1 mM to about 200 mM, about 2 mM to about 100 mM, about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 20 mM L-histidine / L-histidine monohydrochloride buffer.

[0037]

[0043] In some embodiments, the pharmaceutical composition comprises about 1% (wt / vol) to about 20% (wt / vol), about 2% (wt / vol) to about 15% (wt / vol), 5% (wt / vol) to about 12% (wt / vol), about 6% (wt / vol) to about 10% (wt / vol), or about 8% (wt / vol) sucrose.

[0038]

[0044] In some embodiments, the pharmaceutical composition comprises about 0.001% (wt / vol) to about 0.1% (wt / vol), about 0.002% (wt / vol) to about 0.08% (wt / vol), 0.005% (wt / vol) to about 0.06% (wt / vol), about 0.008% (wt / vol) to about 0.04% (wt / vol), about 0.01% (wt / vol) to about 0.03% (wt / vol), or about 0.02% (wt / vol) polysorbate-80.

[0039]

[0045] In some embodiments, the pharmaceutical composition comprises the multifunctional molecule at a concentration of about 0.5 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, about 2 mg / mL to about 80 mg / mL, about 4 mg / mL to about 50 mg / mL, about 6 mg / mL to about 20 mg / mL, about 8 mg / mL to about 12 mg / mL, or about 10 mg / mL.

[0040]

[0046] In some embodiments, the pharmaceutical composition comprises one or more of L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

[0047] In some embodiments, the multifunctional molecule comprises a first polypeptide, a second polypeptide, and a third polypeptide; the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous, and the first polypeptide comprises a first portion of a dimerization module linked to the first portion of the TCRβV6 binding moiety comprising the VH of the TCRβV6 binding moiety; the second polypeptide comprises a second portion of the dimerization module, and IL-2, or a functional fragment or functional variant thereof, is covalently linked to the second polypeptide; and the third polypeptide comprises the second portion of the TCRβV6 binding moiety comprising the VL of the TCRβV6 binding moiety.

[0041]

[0048] In some embodiments, the first polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3517, 4000, 4004, 4006, 4008, 4010, 4011, 4014, 4016, and 4018; the second polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3521, 4002, 4007, 4003, 4013, and 4015; and the third polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3518, 4005, 4009, 4012, and 4017.

[0042]

[0049] In some embodiments, the multifunctional molecule comprises a first polypeptide and a second polypeptide; the first polypeptide and the second polypeptide are discontinuous, the TCRβV6 binding portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single-domain antibody, the first polypeptide comprises a first portion of a dimerization module linked to the TCRβV6 binding portion; the second polypeptide comprises a second portion of the dimerization module, and IL-2, or a functional fragment or functional variant thereof, is covalently linked to the second polypeptide.

[0043]

[0050] In some embodiments, the first polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4019, 4021, 4023, 4025, and 4027, and the second polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4020, 4022, 4024, 4026, and 4028.

[0044] Incorporation by Reference

[0051] All publications, patents, and patent applications mentioned in this specification are herein 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.

[0045]

[0052] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0046] [Figure 1]

[0053] FIG. 1 shows an overview of the treatment and evaluation plan for this study. [Figure 2]

[0054] Figure 1 shows the predicted effect of Compound 1 on Vβ6 / Vβ10 CD8+ T cell expansion (frequency) in humans (70 kg body weight) after a single 1-hour IV infusion across various dose levels. Solid line - median, shaded area - 5th and 95th percentiles from 1000 simulated individuals, dotted line - 20% increase from baseline. [Figure 3]

[0055] FIG. 1 shows a consolidated summary of Compound 1 pharmacology, MABEL estimates, and Cmax predictions for Phase 1 dose escalation. MABEL estimate 1: First diamond from left: EC20 for in vitro human Vβ6 / V10 CD8+ T cell expansion (0.7 nM), and second diamond from left: Cmax at the ED20 dose modeled from an IV monkey study (0.91 nM). MABEL estimate 2: Predicted human Cmax (7.98 nM) for a starting dose of 0.04 mg / kg, predicting a 20% increase in Vβ6 / V10 CD8+ T cell expansion over baseline in ≥95% of patients. *In a repeat-dose GLP toxicology study, moderate hunched posture, reduced activity, and decreased appetite were observed in monkeys administered 1 mg / kg IV Compound 1 for 4-5 days after injection, and in a single-dose non-GLP pharmacology study, fever, diarrhea, and mild dehydration were observed in monkeys administered 1.5 mg / kg Compound 1. These signs resolved by day 8-9 after administration. In a repeat-dose non-GLP monkey pharmacology study, sporadic and transient hunched posture and reduced activity were observed at the 0.5 mg / kg dose level. [Figure 4]

[0056] FIG. 1 shows the Phase 1 study design. [Figure 5]

[0057] This shows that a multifunctional molecule containing an anti-TCRVβ6 binding domain and an IL-2 domain (compound 1) increases TCR signaling as measured by pERK levels compared to a multifunctional molecule containing a non-TCR binding domain and an IL-2 control and a multifunctional molecule containing two non-TCR binding domain controls. [Figure 6-1]

[0058] FIG. 6 shows potent single-agent activity of mSTAR murine surrogate bispecific antibodies (BsAbs) with long-lasting responses in various tumor models, including PD-1 refractory models. [Figure 6-2] FIG. 6 shows potent single-agent activity of mSTAR murine surrogate bispecific antibodies (BsAbs) with long-lasting responses in various tumor models, including PD-1 refractory models. [Figure 6-3] FIG. 6 shows potent single-agent activity of mSTAR murine surrogate bispecific antibodies (BsAbs) with long-lasting responses in various tumor models, including PD-1 refractory models. [Figure 7]

[0059] FIG. 1 shows that mSTAR produces potent tumor regression in the EMT6 model. [Figure 8A]

[0060] Figures 8A-8B show that mSTAR remodels the expansion of tumor-infiltrating lymphocytes (TILs), such as VβCD8+ / CD4+ T effector memory (TEM) cells and central memory T (TCM) cells. Figure 8A shows scRNAseq analysis of EMT6 TILs. Figure 8B shows scRNAseq analysis of TIL subtypes. [Figure 8B] Figures 8A-8B show that mSTAR remodels the expansion of tumor-infiltrating lymphocytes (TILs), such as VβCD8+ / CD4+ T effector memory (TEM) cells and central memory T (TCM) cells. Figure 8A shows scRNAseq analysis of EMT6 TILs. Figure 8B shows scRNAseq analysis of TIL subtypes. [Figure 9]

[0061] Figure 9 shows that mSTAR induces a novel TEM phenotype. For each violin diagram, vehicle is on the left and mSTAR is on the right. [Figure 10A]

[0062] Figures 10A-10B show that mSTAR induces an increase in TCR diversity in TILs. Figure 10A shows that mSTAR increases Vβ TIL clonal diversity. Figure 10B shows a large increase in unique CDR3 transcripts in mSTAR-treated TILs. [Figure 10B] Figures 10A-10B show that mSTAR induces an increase in TCR diversity in TILs. Figure 10A shows that mSTAR increases Vβ TIL clonal diversity. Figure 10B shows a large increase in unique CDR3 transcripts in mSTAR-treated TILs. [Figure 11]

[0063] FIG. 11 shows that Compound 1 induced an expansion of Vβ6CD8+ T cells in monkey blood with minimal Treg. [Figure 12]

[0064] FIG. 12 shows that Compound 1 induces ex vivo expansion of patient TILs and killing of refractory autologous tumors compared to pembrolizumab. [Figure 13]

[0065] FIG. 1 shows that mSTAR promotes "functional memory" and long-term protection as a result of VβCD8+ T cells. [Figure 14]

[0066] FIG. 1 shows that Compound 1 and multifunctional molecules containing a non-TCR binding domain and an IL-2 control increase IL-2R signaling compared to an isotype control, as measured by pSTAT5 levels. [Figure 15]

[0067] Figure 1 shows the prevalence of Vβ6TCR T cells in isolated TILs and PBMCs from cancer patients (n=43) and healthy donors (n=20). For each cancer type, the left bar represents Vβ6-5+ TILs and the right bar represents Vβ6-5+ PBs. [Figure 16]

[0068] 1 is a schematic diagram depicting an exemplary embodiment of a multifunctional molecule comprising a TCRβV-binding moiety and a cytokine polypeptide (e.g., IL2) described herein. [Figure 17]

[0069] FIG. 1 shows that Compound 1 bound to human CD4+ and CD8+ T cells similarly to the single-arm anti-Vβ6 / Vβ10 control. [Figure 18]

[0070] FIG. 1 shows the biological reactivity of IL-2 as pSTAT5 activity across Compound 1, RSV-IL2, and rhIL2. [Figure 19]

[0071] Figure 1 shows a series of FACS plots demonstrating pan T cells or Vβ6-5-sorted T cells with high CD25 levels (CD25Hi) or low CD25 levels (CD25Lo). Pan T cells and Vβ6-5-sorted T cells were expanded using anti-CD3 / CD28 beads supplemented with recombinant human IL-2. An aliquot of expanded Vβ6-5+ T cells was stained for Vβ6-5 and purity was confirmed using PE anti-Vβ6-5. Aliquots of expanded pan T cells and Vβ6-5 T cells showing CD25hi expression were rested for 3 days to allow CD25 downregulation for a phenotype with CD25lo expression. [Figure 20]

[0072] FIG. 1 shows that in stimulated and unstimulated sorted T cell populations (pan T cells or Vβ6-5 sorted T cells containing either high or low levels of CD25 after stimulation with anti-CD3 / CD28 or resting cells, respectively), Compound 1 bound in a Vβ TCR-dependent manner with greater avidity to Vβ6CD25Hi and CD25Lo T cells. [Figure 21]

[0073] Figure 1 shows gene expression analysis chart of TRBV-specific T cell lines P12-Ichikawa and HSB-2 via Nanostring and CD25 expression via FACS. [Figure 22]

[0074] FIG. 1 shows the dose-dependent binding of Compound 1 to P12-Ichikawa and HSB-2 T cell lines. [Figure 23]

[0075] FIG. 1 shows a pie plot of the relative frequencies (n=3) of human T cell Vβ6 (purple) and Vβ10 (blue) transcripts before and after stimulation with Compound 1. [Figure 24]

[0076] Figure 1 shows in vitro TCR sequencing. PBMCs were incubated with Compound 1 for 5 days, and T cells were sequenced for the TCR β chain V (TRBV) gene. Compared to unstimulated T cells (gray), Compound 1 selectively expanded T cells bearing TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-5, and TRBV10-3. (n=3 independent donors). [Figure 25]

[0077] FIG. 1 shows a series of FACS plots showing the expansion of Vβ6 / Vβ10 T cells over an 8 day period. [Figure 26]

[0078] FIG. 1 shows a series of graphs displaying activation of CD4+ and CD8+ T cells as assessed by CD25 expression following stimulation with Compound 1 in solution, or an anti-RSV Fab x IL2 control, or an anti-Vβ6 / Vβ10 control. [Figure 27]

[0079] Figure 1 shows direct cell counts of CD4+ and CD8+ T cells after stimulation with Compound 1. Mean ± SEM, n=4. [Figure 28]

[0080] 1 shows purified T cells incubated with Compound 1 and competing concentrations of soluble IL2R, Vβ6-5 antigen, or a mixture of both. Addition of competitor elicited a dose-dependent inhibition of T cell activation. [Figure 29]

[0081] Figure 1 shows a series of FACS plots demonstrating the differentiation of Vβ6 / Vβ10 CD8+ T cells mediated by Compound 1 (10 nM) compared to isotype and control (RSV-IL2 and anti-Vβ6 / Vβ10). The first column represents Vβ6 / Vβ10 T cells, the middle column represents naive T cells (CD95-), and the right column represents central memory. Asterisks indicate plate binding. Treatment with Compound 1 or control was continued for 7 days before analysis. [Figure 30]

[0082] Figure 24 shows a series of graphs depicting a summary of the analysis of Figure 23 for CD4+ (left) and CD8+ (right) Vβ6 / Vβ10 central memory T cells. Squares represent PBMCs, n=3, circles represent purified T cells, n=2. [Figure 31A]

[0083] Figures 31A-31C show the evaluation of TCR and IL-2R signaling using phospho-SLP76, phospho-ERK, and phospho-STAT5 quantification. Figure 31A shows that Compound 1 increased pSLP76 levels in purified CD8+ T cells compared to those from control molecules. Figure 31B shows that Compound 1 increased TCR signaling compared to single-arm controls, as measured by pERK levels. Figure 31C shows that Compound 1 and IL-2 control increased IL-2 signaling compared to anti-TCRVβ6 / Vβ10 monovalent antibodies, as measured by STAT5 levels. [Figure 31B] Figures 31A-31C show the evaluation of TCR and IL-2R signaling using phospho-SLP76, phospho-ERK, and phospho-STAT5 quantification. Figure 31A shows that Compound 1 increased pSLP76 levels in purified CD8+ T cells compared to those from control molecules. Figure 31B shows that Compound 1 increased TCR signaling compared to single-arm controls, as measured by pERK levels. Figure 31C shows that Compound 1 and IL-2 control increased IL-2 signaling compared to anti-TCRVβ6 / Vβ10 monovalent antibodies, as measured by STAT5 levels. [Figure 31C]Figures 31A-31C show the evaluation of TCR and IL-2R signaling using phospho-SLP76, phospho-ERK, and phospho-STAT5 quantification. Figure 31A shows that Compound 1 increased pSLP76 levels in purified CD8+ T cells compared to those from control molecules. Figure 31B shows that Compound 1 increased TCR signaling compared to single-arm controls, as measured by pERK levels. Figure 31C shows that Compound 1 and IL-2 control increased IL-2 signaling compared to anti-TCRVβ6 / Vβ10 monovalent antibodies, as measured by STAT5 levels. [Figure 32]

[0084] 1 is a bar graph showing the percentage of CD8+ T cells triple positive for CD25, IFNγ, and Granzyme B after treatment with no treatment, Compound 1, or a control. [Figure 33]

[0085] Figure 1 shows activation of mouse splenocytes cultured with dose titrations of mSTAR, RSV-IL2, and isotype control. Top: CD4+ T cells. Bottom: CD8+ T cells. Data represent n=1 of 3 independent donors. [Figure 34]

[0086] FIG. 1 shows the pharmacokinetic profile of mSTAR after a single 0.5, 1.0, or 1.5 mg / kg IP dose in mice. [Figure 35]

[0087] FIG. 1 shows the pharmacodynamic profile of the Vβ13-2 / 3 subset of CD8+ and CD4+ cells and total Tregs after a single 1 mg / kg IP dose in mice. [Figure 36]

[0088]

[0023] Figure 1 shows the biodistribution of mSTAR in tumor, spleen, liver, kidney, and lung tissues of BALB / c EMT6 tumor-bearing mice. Biodistribution was measured 6, 24, 48, 72, and 120 hours after administration of 1 mg / kg mSTAR. Data are shown as mean + / - SEM. [Figure 37]

[0089] FIG. 1 shows the expansion of Vβ13 T cells in mice after three doses of mSTAR, 0.5-1.5 mg / kg IP, but not after vehicle or rhIL-2. [Figure 38A]

[0090] Figures 38A-38C show the levels of perivascular leukocyte infiltration in mice administered rhIL2, PBS, or different concentrations of mSTAR. Figure 38A shows IHC staining of lung and liver tissues showing perivascular leukocyte infiltration. Figure 38B shows quantification of perivascular CD8+ T cells from liver tissue. Figure 38C shows quantification of perivascular CD8+ T cells from lung tissue. [Figure 38B] Figures 38A-38C show the levels of perivascular leukocyte infiltration in mice administered rhIL2, PBS, or different concentrations of mSTAR. Figure 38A shows IHC staining of lung and liver tissues showing perivascular leukocyte infiltration. Figure 38B shows quantification of perivascular CD8+ T cells from liver tissue. Figure 38C shows quantification of perivascular CD8+ T cells from lung tissue. [Figure 38C] Figures 38A-38C show the levels of perivascular leukocyte infiltration in mice administered rhIL2, PBS, or different concentrations of mSTAR. Figure 38A shows IHC staining of lung and liver tissues showing perivascular leukocyte infiltration. Figure 38B shows quantification of perivascular CD8+ T cells from liver tissue. Figure 38C shows quantification of perivascular CD8+ T cells from lung tissue. [Figure 39A]

[0091] Figures 39A-39D show changes in serum liver enzyme markers in mice administered rhIL2, PBS, or mSTAR: Figure 39A shows aspartate aminotransferase, Figure 39B shows alanine transaminase, Figure 39C shows alkaline phosphatase, and Figure 39D shows albumin. [Figure 39B]Figures 39A-39D show changes in serum liver enzyme markers in mice administered rhIL2, PBS, or mSTAR: Figure 39A shows aspartate aminotransferase, Figure 39B shows alanine transaminase, Figure 39C shows alkaline phosphatase, and Figure 39D shows albumin. [Figure 39C] Figures 39A-39D show changes in serum liver enzyme markers in mice administered rhIL2, PBS, or mSTAR: Figure 39A shows aspartate aminotransferase, Figure 39B shows alanine transaminase, Figure 39C shows alkaline phosphatase, and Figure 39D shows albumin. [Figure 39D] Figures 39A-39D show changes in serum liver enzyme markers in mice administered rhIL2, PBS, or mSTAR: Figure 39A shows aspartate aminotransferase, Figure 39B shows alanine transaminase, Figure 39C shows alkaline phosphatase, and Figure 39D shows albumin. [Figure 40A]

[0092] Figures 40A-40B show the effect of mSTAR at various doses on tumor volume measurement and mouse survival. Increasing concentrations of mSTAR were administered IP once weekly per four treatments. Triangles indicate dosing intervals. n=8 / group, ****p<0.0001, ***p<0.001, **p<0.01, ns=not significant. Figure 40A shows that mSTAR produced potent tumor regression at 0.3 mg / kg, 0.5 mg / kg, and 1.0 mg / kg in the EMT6 model. Figure 40B shows potent single-agent activity of mSTAR at 1.0 mg / kg in the EMT6 model, with a durable response. [Figure 40B]Figures 40A-40B show the effect of mSTAR at various doses on tumor volume measurement and mouse survival. Increasing concentrations of mSTAR were administered IP once weekly per four treatments. Triangles indicate dosing intervals. n=8 / group, ****p<0.0001, ***p<0.001, **p<0.01, ns=not significant. Figure 40A shows that mSTAR produced potent tumor regression at 0.3 mg / kg, 0.5 mg / kg, and 1.0 mg / kg in the EMT6 model. Figure 40B shows potent single-agent activity of mSTAR at 1.0 mg / kg in the EMT6 model, with a durable response. [Figure 41A]

[0093] Figures 41A-41B show the effect of a single dose of mSTAR at 1.0 mg / kg on tumor volume measurement and mouse survival. Triangles indicate time of administration. n=8 / group, **p<0.01, *p<0.05, ns=not significant. Figure 41A shows that mSTAR produced potent tumor regression in the EMT6 model. Figure 41B shows the potent activity of single dose mSTAR with a long-lasting response in the EMT6 model. [Figure 41B] Figures 41A-41B show the effect of a single dose of mSTAR at 1.0 mg / kg on tumor volume measurement and mouse survival. Triangles indicate time of administration. n=8 / group, **p<0.01, *p<0.05, ns=not significant. Figure 41A shows that mSTAR produced potent tumor regression in the EMT6 model. Figure 41B shows the potent activity of single dose mSTAR with a long-lasting response in the EMT6 model. [Figure 42]

[0094] Figure 1 shows tumor growth curves for mice treated with mSTAR. Studies were performed on randomized mice with tumor volumes of 80-150 mm3. For all models except RM1, mice were dosed at 1 mg / kg once weekly (QW) for 3-4 weeks, and survival was determined based on a tumor volume endpoint of 2000 mm3. For RM1, mice were given 1.5 mg / kg twice weekly (2QW). [Figure 43]

[0095] Figure 1 shows Kaplan-Meier survival curves for treated mice. Studies were performed on randomized mice with tumor volumes of 80-150 mm3. For all models except RM1, mice were dosed at 1 mg / kg once weekly (QW) for 3-4 weeks, and survival was determined based on a tumor volume endpoint of 2000 mm3. For RM1, mice were given 1.5 mg / kg twice weekly (2QW). [Figure 44]

[0096] Figure 1 shows Kaplan-Meier survival curves for treated mice. Studies were performed on randomized mice with tumor volumes of 80-150 mm3. For MC38 and Renca tumor models, mice treated with mSTAR were administered a dosage of 1 mg / kg once weekly for 3 weeks. For RM1, mice received 1.5 mg / kg once weekly. For anti-PD1, mice received 10 mg / kg anti-mouse PD1 twice weekly for a total of 5 treatments. Survival was determined based on a tumor volume endpoint of 2000 mm3. [Figure 45]

[0097] FIG. 1 shows that mSTAR produced potent tumor regression in the EMT6 model compared to single-arm controls (n=8, mean + / - SEM, p<0.0001). [Figure 46]

[0098] FIG. 1 shows IHC staining of EMT6 tumors for CD8 and granzyme B expression. [Figure 47]

[0099] Immunophenotyping of CD8+ TILs isolated from EMT6 mice after tumor implantation. Mean ± SEM, n=4. ****p<0.0001, ***p<0.001, **p<0.01, ns=not significant. [Figure 48]

[0100] Immunotyping of NK and B cells isolated from EMT6 mice after tumor implantation. Mean ± SEM, n = 4. ns = not significant. [Figure 49]

[0101] Immunophenotyping of non-CD8+ TILs isolated from EMT6 mice after tumor implantation. Mean ± SEM, n=4. ***p<0.001, **p<0.01, *p<0.05, ns=not significant. [Figure 50]

[0102] Immunophenotyping of CD8+ TILs isolated from EMT6 mice after tumor implantation comparing RSV F(ab)2×(IL-2)2 and mSTAR treatment. Mean±SEM, n=4. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant. [Figure 51]

[0103] Figure 1 shows that 1.0 mg / kg mSTAR produced potent tumor regression in the EMT6 model. Depletion of Vβ13 T cells abolished the anti-tumor activity of mSTAR. [Figure 52]

[0104] Figure 1 shows the results of a tumor rechallenge study. Left: EMT6 tumors were rejected, whereas CT26 tumors grew, suggesting the establishment of a memory response to EMT6 tumors, possibly mediated via mSTAR treatment. Right: Depletion of CD8+ T cells prior to rechallenge resulted in EMT6 tumor growth. [Figure 53]

[0105] FIG. 10 shows UMAP plots illustrating results from single-cell analysis of EMT6 TIL transcriptome for CD4+ and CD8+ gene expression. [Figure 54]

[0106] Single cell RNAseq analysis of single CD4+ or CD8+ TILs isolated from EMT6 mice at day 14 after tumor implantation following a single dose of mSTAR (right) or vehicle (left) from n=5 pooled mice per group. [Figure 55]

[0107] FIG. 1 shows that Vβ13 T cells were positively labeled based on gene expression of TRBV13-2 and TRBV13-3. [Figure 56]

[0108] FIG. 55 shows Vβ13 T cells across the UMAP plot of FIG. 54 inferred from the expression of TRBV13-2 and TRBV13-3 transcripts. [Figure 57]

[0109] Quantification of cell subsets in EMT6 TILs from mice treated with vehicle or mSTAR. For each cell subset, the top bar represents control (CTRL) and the bottom bar represents mSTAR. [Figure 58]

[0110] FIG. 1 shows quantification of Vβ13 T cell and TIL subtypes. [Figure 59]

[0111] FIG. 10 shows a heatmap illustrating the number of differentially expressed genes (DEGs) in TILs when comparing the Vβ13+ subset from mSTAR with vehicle-treated mice. [Figure 60A]

[0112] Figures 60A-60D show a series of volcano plots of differentially expressed genes between the Vβ13 T cell subset targeted by mSTAR treatment and the group treated with vehicle control. [Figure 60B] Figures 60A-60D show a series of volcano plots of differentially expressed genes between the Vβ13 T cell subset targeted by mSTAR treatment and the group treated with vehicle control. [Figure 60C] Figures 60A-60D show a series of volcano plots of differentially expressed genes between the Vβ13 T cell subset targeted by mSTAR treatment and the group treated with vehicle control. [Figure 60D] Figures 60A-60D show a series of volcano plots of differentially expressed genes between the Vβ13 T cell subset targeted by mSTAR treatment and the group treated with vehicle control. [Figure 61]

[0113]

[0023] Figure 1 shows a heatmap of differentially expressed genes in response to mSTAR treatment compared to vehicle across the indicated T cell subsets. Expression values ​​are scaled for each gene. [Figure 62]

[0114] Figure 1 shows that mSTAR induced a novel CD8-TEM phenotype. For each plot, the left violin represents vehicle and the right violin represents mSTAR. [Figure 63]

[0115] FIG. 1 shows a series of heat maps of differentially expressed genes identified separately from mSTAR treatment compared to IL-2, anti-PD-1, and anti-PD-1-IL-2 mutein treatments from published studies. [Figure 64]

[0116] FIG. 1 shows a series of Venn diagrams showing the number of overlapping genes between distinct Compound 1 genes and genes significantly differentially expressed from vehicle and IL-2, anti-PD-1 and anti-PD-1-IL-2 mutein treatments. [Figure 65]

[0117] FIG. 1 shows a series of violin diagrams depicting TCR signaling repressor genes after treatment in CD8 effector T cell subsets and CD8' superior effector' T cells from published studies. [Figure 66]

[0118] Figure 1 shows clonal diversity within each TRBV gene from TILs obtained from EMT6 mice treated with vehicle (top) or mSTAR (bottom). [Figure 67]

[0119] Figure 1 shows that mSTAR induced an increase in TCR diversity in TILs. Top: Treatment with mSTAR increased clonal diversity in targeted Vβ13 T cells, but not in untargeted Vβ5 T cells. Bottom: Bubble plots show a large increase in unique CDR3 transcripts in mSTAR-treated Vβ13 TILs compared to those treated with vehicle. [Figure 68]

[0120] FIG. 1 shows quantification of clonal diversity of TILs between vehicle and mSTAR-treated mice using the inverse of Simpson's index. [Figure 69]

[0121] FIG. 1 shows single-cell RNAseq analysis of Vβ13 and Vβ5 T cells in vehicle- and mSTAR-treated mice. [Figure 70]

[0122] FIG. 1 shows IFN-γ intracellular FACS staining from an ex vivo tumor antigen recall assay in splenocytes, Vβ13CD8+ T cells isolated from EMT6 tumor-bearing mice treated with 0, 0.5, 1, or 1.5 mg / kg mSTAR. [Figure 71]

[0123] Figure 1 shows clonal diversity in TILs among MC38 mice treated with vehicle, mSTAR, or anti-RSV-IL-2. Left: clone size. Right: inverse Simpson's index for diversity. [Figure 72]

[0124] FIG. 72 shows similar data to FIG. 71 in CT26 tumor mice. [Figure 73]

[0125] FIG. 1 shows isolated tumor-infiltrating lymphocytes (TILs) from mSTAR-treated CT26 tumor-bearing mice stained for tetramers recognizing the tumor rejection antigen AH1 / gp70 in Vβ13+CD8+ and Vβ13-CD8+ T cells. [Figure 74]

[0126] FIG. 1 shows the pharmacokinetic profile (serum concentration over time) of a single IV dose of Compound 1 in cynomolgus monkeys. [Figure 75]

[0127] Figure 1 shows T cell frequencies in blood after a single IV dose of 1 mg / kg Compound 1. n=3 monkeys. [Figure 76]

[0128] Figure 1 shows serum soluble CD25 levels in monkeys administered a single IV dose of 0.5 mg / kg Compound 1. Mean ± SEM, n=6. [Figure 77]

[0129] Figure 1 shows serum levels of IFNγ, TNFα, and IL-6 in monkeys administered a single IV dose of 0.5 mg / kg Compound 1. Mean ± SEM, n=6. [Figure 78]

[0130] Figure 1 shows serum levels of IL-5 and eosinophil counts in monkeys administered a single IV dose of 1 mg / kg Compound 1. Mean ± SEM, n=3. [Figure 79]

[0131] Figures 79A-79D show serum levels of liver enzyme markers in monkeys after a single IV dose of Compound 1. Figure 79A shows aspartate aminotransferase, Figure 79B shows alanine transaminase, Figure 79C shows alkaline phosphatase, and Figure 79D shows albumin. [Figure 80]

[0132]

[0023] Figure 1 shows that Compound 1 induced ex vivo expansion of patient TILs and killing of refractory autologous tumors compared to pembrolizumab. Autologous T cells were incubated with 3 μg / ml Compound 1, 10 μg / ml pembrolizumab, or 3 μg / ml isotype control for 5 days. [Figure 81]

[0133] FIG. 1 shows the frequency of Vβ6 / Vβ10 T cells across four organoid models. [Figure 82]

[0134] Figure 1 shows the lethality of human tumor organoids mediated by Compound 1, which are generated from primary patient-derived tissues from colorectal and NSCLC cancer patients. Vertical bars represent the percentage of organoid area reduced after incubating organoids with Compound 1 and autologous TILs compared to isotype control. Mean ± SEM, n=4, ****p<0.0001, **p<0.01, ns=not significant. [Figure 83]

[0135] Figure 1 shows dose-dependent cancer organoid killing in NSCLC PDX models using Compound 1. No reduction in organoid size was observed for the IL-2 control molecule. [Figure 84]

[0136] Figure 1 shows Compound 1-mediated ex vivo activation of HPV-16-specific T cells in PBMCs from healthy donors. Mean ± SEM, n=7, *p<0.05, ns=not significant. PBMCs were treated with 1 nM Compound 1, isotype control, or medium for 1 hour, then stimulated with HPV-16 peptide or negative control and stained for intracellular expression of IFNγ, TNFα, IL-2, and CD107a. [Figure 85]

[0137] Figure 1 shows ex vivo expansion of HPV-16 targeting tumor antigen-specific T cells mediated by Compound 1. PBMCs were obtained from healthy donors and treated with 1 nM Compound 1, anti-TCRVβ6 / Vβ10 antibody, isotype control, or medium for 1 hour, then stimulated with a 15-mer peptide overlapping HPV-16 or a negative control for 7 days. [Figure 86]

[0138] Figure 1 shows Compound 1-mediated ex vivo activation of HPV-16-specific T cells in PBMCs from cervical cancer patients. PBMCs were treated with 1 nM Compound 1, isotype control, or medium for 1 hour, then stimulated with HPV-16 peptide or negative control and stained for intracellular expression of IFNγ, TNFα, IL-2, and CD107a. [Figure 87A]

[0139] Figures 87A-87B show two SDS-PAGE gels and size exclusion chromatography analysis plots for Compound 1 (Figure 87A) and mSTAR (Figure 87B). [Figure 87B] Figures 87A-87B show two SDS-PAGE gels and size exclusion chromatography analysis plots for Compound 1 (Figure 87A) and mSTAR (Figure 87B). DETAILED DESCRIPTION OF THE INVENTION

[0047] definition

[0140] Certain specific details of the present description are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0048]

[0141] Unless the context requires otherwise, throughout this specification and the claims that follow, the word "comprise" and variations thereof, such as "comprises" and "comprising," should be interpreted in the open-ended, inclusive sense of "including but not limited to." Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0049]

[0142] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. The terms "a" or "an," when used in conjunction with the word "comprising," may mean "one," but are also consistent with the meanings of "one or more," "at least one," and "one or more."

[0050]

[0143] It should be noted that the term "or" is also generally used to mean "and / or" unless the context dictates otherwise.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0051]

[0145] The term "about," when referring to a measurable value such as an amount, a duration of time, and the like, is meant to encompass a ±20% variation, or in some cases a ±10% variation, or in some cases a ±5% variation, or in some cases a ±1% variation, or in some cases a ±0.1% variation, where such variations are appropriate for practicing the methods of the present disclosure. As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values.

[0052]

[0146] The terms "obtain" or "obtaining," as the terms are used herein, refer to obtaining a physical entity (e.g., a sample, polypeptide, nucleic acid, or sequence) or a value, e.g., a numerical value, by "directly obtaining" or "indirectly obtaining" the physical entity or value. "Directly obtaining" refers to performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a physical entity includes performing a process that involves a physical change in a physical substance, e.g., a starting material. Directly obtaining a value includes performing a process that involves a physical change in a sample or other substance, e.g., performing an analytical process that involves a physical change in a substance, e.g., a sample.

[0053]

[0147] As used herein, "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that contains at least one immunoglobulin variable domain structure and / or sequence. Antibody molecules encompass antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that occurs naturally or is formed by conventional immunoglobulin gene fragment recombination processes. In embodiments, an antibody molecule refers to an immunologically active antigen-binding portion of an immunoglobulin molecule, e.g., an antibody fragment. An antibody fragment, e.g., a functional fragment, is a portion of an antibody, e.g., Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). A functional antibody fragment binds to the same antigen recognized by an intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable regions, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, or a recombinant single-chain polypeptide molecule ("scFv protein") in which the light and heavy chain variable regions are connected by a peptide linker. In some embodiments, an antibody fragment does not include a portion of an antibody that does not have antigen-binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, such as dAb (domain antibodies), single-chain, Fab, Fab', and F(ab')2 fragments, and single-chain variable fragments (scFv). In some embodiments, the antibody molecule is an antibody mimetic. In some embodiments, the antibody molecule is or includes an antibody-like framework or scaffold, such as fibronectin, ankyrin repeats (e.g., designed ankyrin repeat proteins (DARPins)), avimers, affibodies, affinity ligands, anticalins, or affilin molecules.

[0054]

[0148] The term "human-like antibody molecule," as used herein, refers to a humanized antibody molecule, a human antibody molecule, or an antibody molecule having at least 95% sequence identity with non-murine germline framework regions, e.g., FR1, FR2, FR3, and / or FR4. In some embodiments, a human-like antibody molecule comprises a framework region having at least 95% sequence identity with a human germline framework region, e.g., FR1, FR2, FR3, and / or FR4 of a human germline framework region. In some embodiments, a human-like antibody molecule is a recombinant antibody. In some embodiments, a human-like antibody molecule is a humanized antibody molecule. In some embodiments, a human-like antibody molecule is a human antibody molecule. In some embodiments, a human-like antibody molecule is a phage-displayed or yeast-displayed antibody molecule. In some embodiments, a human-like antibody molecule is a chimeric antibody molecule. In some embodiments, a human-like antibody molecule is a CDR-grafted antibody molecule.

[0055]

[0149] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other alterations that are compatible with forming a protein structure.

[0056]

[0150] In embodiments, an antibody molecule is monospecific, e.g., comprises binding specificity for a single epitope. In some embodiments, an antibody molecule is multifunctional, e.g., comprises multiple immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, an antibody molecule is a bispecific antibody molecule. A "bispecific antibody molecule," as used herein, refers to an antibody molecule that has specificity for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0057]

[0151] As used herein, "antigen" (Ag) refers to a molecule capable of eliciting an immune response, e.g., involving activation of certain immune cells and / or antibody production. Any macromolecule can be an antigen, including almost any protein or peptide. Antigens can also be derived from genome recombinants or DNA. For example, any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein capable of eliciting an immune response encodes an "antigen." In embodiments, an antigen need not be encoded solely by the full-length nucleotide sequence of a gene, or even by a gene at all. In embodiments, antigens can be synthesized or derived from a biological sample, e.g., a tissue sample, a tumor sample, cells, or a fluid associated with other biological components. As used herein, "tumor antigen" or, interchangeably, "cancer antigen" includes any molecule present in or associated with the microtubule environment of a cancer, e.g., a cancer cell or tumor, that can elicit an immune response. As used herein, "immune cell antigen" includes any molecule present in or associated with an immune cell that can elicit an immune response.

[0058]

[0152] The "antigen-binding site," or "binding portion," of an antibody molecule refers to the portion of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding. In embodiments, the antigen-binding site is formed by amino acid residues in the variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains are called hypervariable regions and are located between adjacent, more conserved stretches called "framework regions" (FRs). FRs are amino acid sequences naturally found adjacent to and between hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." Framework regions and CDRs are defined and described, for example, in Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0059]

[0153] As used herein, "immune cell" refers to any of a variety of cells that function in the immune system, for example, to protect against infectious pathogens or foreign substances. In embodiments, the term includes white blood cells, such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate white blood cells include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate white blood cells identify and eliminate pathogens by either attacking larger pathogens through contact or engulfing and subsequently killing microorganisms, and are mediators in the activation of adaptive immune responses. Cells of the adaptive immune system are a specialized type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in humoral immune responses, while T cells are involved in cell-mediated immune responses. The term "immune cell" includes immune effector cells.

[0060]

[0154] "Immune effector cells," as that term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0061]

[0155] The term "effector function" or "effector response" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including the secretion of cytokines.

[0062]

[0156] The terms "polypeptide," "peptide," and "protein" (when single-chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant technology from eukaryotic or prokaryotic hosts, or can be the product of synthetic procedures.

[0063]

[0157] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can be single- or double-stranded, and a single strand can be the coding strand or the non-coding (antisense) strand. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide that is not naturally occurring or that is linked to another polynucleotide in a non-natural configuration, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin.

[0064]

[0158] The term "isolated" as used herein refers to a material that has been removed from its original or natural environment (e.g., the natural environment in which it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is still isolated in the sense that it is not part of the environment in which it is found in nature. An isolated polynucleotide (ribonucleic acid (RNA), deoxyribonucleic acid (DNA)) or polypeptide does not contain the adjacent genes / nucleic acids or sequences / amino acids with which it occurs in its naturally occurring state.

[0065]

[0159] The compositions and methods of the present invention encompass polypeptides and nucleic acids having a designated sequence or a sequence substantially identical or similar thereto, e.g., a sequence at least 80%, 85%, 90%, 95%, or more identical to the designated sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences may share a common structural domain and / or common functional activity. For example, the amino acid sequence may contain a common structural domain that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with a reference sequence, e.g., a sequence provided herein. In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleotide sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or common functional activity. For example, a nucleotide sequence has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, such as a sequence provided herein.

[0066]

[0160] The term "variant" refers to a polypeptide having an amino acid sequence substantially identical to, or encoded by a nucleotide sequence substantially identical to, a reference amino acid sequence. In some embodiments, the variant is a functional variant. In some embodiments, the TCRβ variant is capable of binding to TCRα to form a TCRα:β complex.

[0067]

[0161] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and that is capable of possessing one or more activities of the reference amino acid sequence.

[0068]

[0162] Calculation of homology or sequence identity (the terms are used interchangeably herein) between sequences is performed as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

[0069]

[0163] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.Comparing sequences between two sequences and determining percent identity can be achieved using a mathematical algorithm.In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) is the Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0070]

[0164] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as "query sequences" to perform searches against public databases to identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0071]

[0165] It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions which do not substantially affect their function.

[0166] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functionalities and can be included in naturally occurring amino acid polymers. Exemplary amino acids include naturally occurring amino acids, their analogs, analogs, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D- or L-engineered isomers and peptidomimetics.

[0072]

[0167] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0073]

[0168] As used herein, the term "molecule," e.g., when used in antibody molecules, cytokine molecules, receptor molecules, includes full-length naturally occurring molecules as well as variants, e.g., functional variants (e.g., truncations, fragments, mutations (e.g., substantially similar sequences) or derivative forms thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule is retained.

[0074]

[0169] As used herein, the term "mutation" refers to a change in the nucleotide sequence of an organism, a virus genome, or extrachromosomal DNA. In some embodiments, the mutation can be a large-scale mutation, such as an amplification (or gene duplication), or a repeat of a chromosomal segment, a deletion of a large chromosomal region, a chromosomal rearrangement (e.g., a chromosomal translocation, a chromosomal inversion, a non-homologous chromosomal crossover, and an interstitial deletion), and a loss of heterozygosity. In some embodiments, the mutation can be a small-scale mutation, such as an insertion, a deletion, and a substitution mutation. As used herein, the term "substitution mutation" refers to a transition in which one nucleotide is exchanged for another nucleotide.

[0075]

[0170] "Interleukin-2," as referred to herein, is also known as IL2, IL-2, IL 2, TCGF, ​​lymphokine, and interleukin 2, and includes any recombinant or naturally occurring form of IL-2 or a variant or homolog thereof that has or maintains IL-2 activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity). In some aspects, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to naturally occurring IL-2. In some embodiments, the IL-2 is substantially identical to or a variant or homolog having substantial identity to the protein identified by UniProt reference number P60568.

[0076] Anti-TCRβV antibody Human T cell receptor (TCR) complex

[0171] TCRs are disulfide-linked membrane-bound heterodimeric proteins consisting of highly variable alpha (α) and beta (β) chains, typically expressed as part of a complex with an invariant CD3 chain molecule. The TCR on αβ T cells is formed by a heterodimer of one alpha and one beta chain. Each alpha or beta chain consists of a constant domain and a hypervariable domain, classified as an immunoglobulin superfamily (IgSF) fold. TCRβV chains can be further classified into 30 subfamilies (TRBV1-30). Despite their high structural and functional homology, amino acid sequence homology within TRBV genes is very low. Approximately only 4 of 95 amino acids are identical, with 10 additional amino acids conserved across all subfamilies. Nevertheless, TCRs formed between highly divergent alpha and beta chains exhibit significant structural homology and similar functions, such as inducing T cell activation.

[0077]

[0172] T cell receptors (TCRs) can be found on the surface of T cells. TCRs recognize, for example, bound antigens, such as peptides, presented on major histocompatibility complex (MHC) molecules on the surface of cells, such as antigen-presenting cells. TCRs are heterodimeric molecules and can include an alpha chain, a beta chain, a gamma chain, or a delta chain. TCRs comprising an alpha chain and a beta chain are also referred to as TCRαβ. The TCR beta chain consists of the following regions (also known as segments): variable (V), diversity (D), joining (J), and constant (C) (see Mayer G. and Nyland J. (2010) Chapter 10: Major Histocompatibility Complex and T-cell Receptors—Role in Immune Responses. In: Microbiology and Immunology online, University of South Carolina School of Medicine). The TCR alpha chain consists of V, J, and C regions. Rearrangement of the T cell receptor (TCR) through somatic recombination of the V (variable), D (diversity), J (joining), and C (constant) regions is a distinct event in T cell development and maturation. TCR gene rearrangement occurs in the thymus.

[0078]

[0173] A TCR can comprise a receptor complex, also known as a TCR complex, which comprises a TCR heterodimer comprising an alpha chain and a beta chain, and a dimeric signaling molecule, e.g., a CD3 co-receptor, e.g., CD3δ / ε, and / or CD3γ / ε.

[0079]

[0174] As used herein, the term "T cell receptor beta variable chain" or "TCRβV" refers to the extracellular region of the T cell receptor beta chain that contains the antigen recognition domain of the T cell receptor. The term TCRβV includes mammalian isoforms, such as human TCRβV, human species homologs, and analogs that share at least one epitope with TCRβV. Human TCRβV includes, but is not limited to, TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily, TCRβ V29 subfamily, TCRβ V1 subfamily, TCRβ V1 subfamily, TCRβ V17 subfamily, TCRβ V21 subfamily, TCRβ In some embodiments, the TCRβ V6 subfamily includes a gene family comprising a subfamily that includes a TCRβ V6-4 subfamily, a TCRβ V23 subfamily, or a TCRβ V26 subfamily, as well as family members of the subfamily and variants thereof (e.g., structural or functional variants thereof). * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5 * 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01, or TCRβ V6-1 *In some embodiments, the TCRβ V includes TCRβ V6-5 * 01, or variants thereof, such as variants having 85%, 90%, 95%, 99% or more identity to the naturally occurring sequence. * 01 is also known as TRBV65; TCRBV6S5; TCRBV13S1, or TCRβ V13.1. * 01, e.g., human TCRβ V6-5 * The amino acid sequence of TCRβ V6-5 is known in the art, for example, as provided by IMGT ID L36092. * 01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or more identity thereto. * 01 comprises the amino acid sequence of SEQ ID NO: 44, or a sequence having 85%, 90%, 95%, 99% or more identity thereto.

[0080] Sequence number 43 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACC CAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC SEQ ID NO: 44 MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHY-SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY TCR beta V (TCRβV)

[0175] Diversity of the immune system allows protection against a vast array of pathogens. Because the germline genome is limited in size, diversity is achieved not only through the V(D)J recombination process, but also through junction (junction between VD and DJ segments) deletion of nucleotides and pseudo-random, non-templated addition of nucleotides. TCR beta genes undergo genetic rearrangement to generate diversity.

[0081]

[0176] TCR V beta repertoires vary between individuals and populations due to, for example, seven frequent inactivating polymorphisms in functional gene segments and a large insertion / deletion-associated polymorphism encompassing two V beta gene segments.

[0082]

[0177] Provided herein are, inter alia, antibody molecules and fragments thereof that specifically bind to, for example, a human TCR beta V chain (TCRβV), for example, a TCRβV gene family (also referred to as a group), for example, a TCRβV subfamily (also referred to as a subgroup) described herein. TCR beta V families and subfamilies are known in the art and are described, for example, in Yassai et al., (2009) Immunogenetics 61(7)pp:493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3)pp:201-206. The antibodies described herein may be recombinant antibodies, for example, recombinant non-mouse antibodies, for example, recombinant human or humanized antibodies.

[0083]

[0178] The terms TCRBV, TCRVB, TRBV, TCRβV, TCRVβ, or TRβV are used interchangeably herein and refer to, for example, a TCR beta V chain as described herein.

[0084]

[0179] In some embodiments, provided herein are anti-TCRβ antibody molecules that bind to a human TCRβV, e.g., a TCRβV family, e.g., a gene family or variant thereof. In some embodiments, the TCRβV gene family includes one or more subfamilies, e.g., as described herein, e.g., in Table 8A or Table 8B. In some embodiments, the TCRβV gene family includes the TCRβ V6 subfamily or the TCRβ V10 subfamily.

[0085]

[0180] In some embodiments, the TCRβ V6 subfamily is also known as TCRβ V13.1. In some embodiments, the TCRβ V6 subfamily is also known as TCRβ V6-4 * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5 * 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01, or TCRβ V6-1 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5* 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-2 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01, or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01, or any variant thereof.

[0086]

[0181] In some embodiments, TCRβ V6 is TCRβ V6-5 * 01, or a variant thereof. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized by, e.g., bound by, e.g., SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, TCRβ V6, e.g., TCRβ V6-5 * 01 is recognized by, e.g., bound by, SEQ ID NO:9 and / or SEQ ID NO:10. In some embodiments, TCRβ V6 is recognized by, e.g., bound by, SEQ ID NO:9 and / or SEQ ID NO:11.

[0087]

[0182] In some embodiments, the TCRβ V10 subfamily is TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or any variant thereof.

[0088]

[0183] Exemplary amino acid sequences of TCRβV subfamily members can be found on the ImMunoGeneTics Information System website: http: / / www.imgt.org / , or similar resources.

[0089] Anti-TCRβV antibody

[0184] For cancer immunotherapy, current bispecific constructs designed to redirect T cells to promote tumor cell lysis typically utilize antibody fragments (e.g., Fab, scFv, VH, single-domain antibodies) derived from monoclonal antibodies (mAbs) directed against the CD3e subunit of the T cell receptor (TCR). However, this approach has limitations that may prevent the full realization of the therapeutic potential of such bispecific constructs. Previous studies have shown that even low "activating" doses of anti-CD3e mAbs can cause long-term T cell dysfunction and exert immunosuppressive effects. In addition, anti-CD3e mAbs are associated with side effects due to massive T cell activation. Large numbers of activated T cells secrete substantial amounts of cytokines, the most important of which is interferon gamma (IFNγ). This excess amount of IFNγ then activates macrophages, which then overproduce pro-inflammatory cytokines such as IL-1 beta, IL-6, IL-10, and TNF-alpha, causing a "cytokine storm," also known as cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., J Immunother Cancer. 2018 Jun 15;6(1):56, incorporated herein by reference in its entirety). Thus, there is a need to develop antibodies capable of binding to and activating only a subset of effector T cells, for example, to reduce CRS and / or neurotoxicity (NT).

[0090]

[0185] Described herein are molecules and methods for targeting the TCRβV chain of TCR. Without wishing to be bound by theory, such molecules may bind, activate, and / or expand only a subset of T cells, avoid or reduce CRS and / or NT, and minimize the potential immunosuppressive effects of anti-CD3 mAbs.

[0091]

[0186] Described herein is a class of antibodies, the anti-TCRβV antibody molecules described herein, that recognize structurally conserved but sequence-variable regions, e.g., domains, on the TCRβV protein and have similar functions (e.g., T cell activation and cytokine profiles similar to those described herein), despite having low sequence similarity (e.g., low sequence identity among different antibody molecules that recognize different TCRβV subfamilies). Thus, the anti-TCRβV antibody molecules described herein share a structure-function relationship.

[0092]

[0187] Without wishing to be bound by theory, in some embodiments, the anti-TCRβV antibody molecules described herein bind to an epitope that faces the outside of the TCRβV protein when in a complex with a TCR alpha protein. In some embodiments, the anti-TCRβV antibody molecules described herein recognize (e.g., bind to) a domain (e.g., an epitope) on the TCRβV protein that is (1) structurally conserved among different TCRβV subfamilies; and (2) has minimal sequence identity among different TCRβV subfamilies. TCRβV proteins from different TCRβV subfamilies share minimal sequence similarity. However, TCRβV proteins with minimal sequence similarity share similar 3D conformations and structures.

[0093]

[0188] Alignment of TCR sequences underestimates the diversity of TCR sequences, especially since TCR sequences from different subfamilies are quite different from each other.

[0189] In some embodiments, the anti-TCRβV antibody molecules described herein do not recognize, e.g., do not bind to, the interface of the TCRβV:TCR alpha complex. In some embodiments, the anti-TCRβV antibody molecules described herein do not recognize, e.g., do not bind to, the constant region of the TCRβV protein. An exemplary antibody that binds to the constant region of the TCRβV region is JOVI.1, as described by Viney et al. (Hybridoma. 1992 Dec;11(6):701-13). In some embodiments, the anti-TCRβV antibody molecules described herein do not recognize, e.g., do not bind to, one or more (e.g., all) of the complementarity-determining regions (e.g., CDR1, CDR2, and / or CDR3) of the TCRβV protein.

[0094]

[0190] Provided herein, among other things, are antibody molecules directed against the variable chain of the beta subunit of the TCR (TCRβV), which binds to and, e.g., activates, a subset of T cells. The anti-TCRβV antibody molecules described herein result in reduced or no production of CRS-associated cytokines, e.g., IL-6, IL-1beta, IL-10, and TNFalpha; and enhanced and / or delayed production of IL-2 and IFNγ. In some embodiments, the anti-TCRβV antibody molecules described herein have a cytokine profile that differs from that of T cell engagers that bind to receptors or molecules other than the TCRβV region ("non-TCRβV-binding T cell engagers"), e.g., as described herein. In some embodiments, non-TCRβV-binding T cell engagers include antibodies that bind to CD3 molecules (e.g., CD3 epsilon (CD3e) molecules); or TCR alpha (TCRα) molecules. In some embodiments, the non-TCRβV-binding T cell engager is an OKT3 antibody or an SP34-2 antibody.

[0095]

[0191] In some embodiments, the anti-TCRβV antibody molecules described herein result in the expansion of TCRβV+ T cells, e.g., a subset of effector memory T cells, also known as TEMRA. Without wishing to be bound by theory, in some embodiments, it is believed that TEMRA cells can promote tumor cell lysis but not CRS. Accordingly, methods for producing the anti-TCRβV antibody molecules and uses thereof are provided herein. Similarly, multifunctional molecules, e.g., bispecific molecules comprising the anti-TCRβV antibody molecules, are described herein. In some embodiments, compositions comprising the anti-TCRβV antibody molecules of the present disclosure can be used (1) to activate and redirect T cells to promote tumor cell lysis for cancer immunotherapy; and / or (2) to expand TCRβV+ T cells. In some embodiments, compositions comprising the anti-TCRβV antibody molecules described herein limit the adverse side effects of CRS and / or NT, e.g., CRS and / or NT associated with anti-CD3e targeting.

[0096]

[0192] In some embodiments, the anti-TCRβV antibody molecule binds to one or more of TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, and TRBV6-9. In some embodiments, the anti-TCRβV antibody molecule is anti-TRBV6-1, anti-TRBV6-2, anti-TRBV6-3, anti-TRBV6-4, anti-TRBV6-5, anti-TRBV6-6, anti-TRBV6-8, or anti-TRBV6-9. Exemplary anti-TCRβV antibody molecules and the corresponding TCRβV subfamilies recognized by the anti-TCRβV antibody molecules are disclosed in Table 10A.

[0097]

[0193] In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, or TRBV6-9. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-1. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-2. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-3. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-4. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-5. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-6. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-8. In some embodiments, the anti-TCRβV antibody molecule specifically binds to TRBV6-9.

[0098]

[0194] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The light or heavy chain variable framework of the antibody molecule (e.g., a region encompassing at least FR1, FR2, FR3, and optionally FR4) comprises: (a) a light or heavy chain variable framework that comprises human light or heavy chain variable framework amino acid residues from a human mature antibody, a human germline sequence, or a human consensus sequence, e.g., at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or 100% of the light or heavy chain variable framework residues; (b) a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a light or heavy chain variable framework comprising human light or heavy chain variable framework amino acid residues from a germline sequence or human consensus sequence, e.g., 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the light or heavy chain variable framework residues; (d) a non-human framework (e.g., a rodent framework); or (e.g., a non-human framework that has been modified, e.g., to remove antigenic or cytotoxic determinants, e.g., deimmunized or partially humanized. In some embodiments, the light or heavy chain variable framework region (particularly FR1, FR2, and / or FR3) comprises a light or heavy chain variable framework that is at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, or 99% identical to the framework of the VL or VH segment of a human germline gene.

[0099]

[0195] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain variable domain having at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, or more changes, e.g., amino acid substitutions or deletions, from the amino acid sequence of any one of AH.1 to AH.85, e.g., AH.1, AH.2, or AH.68, e.g., the amino acid sequence of the FR region in the entire variable region set forth in SEQ ID NO: 9.

[0100]

[0196] Alternatively, or in combination with the heavy chain substitutions described herein, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a light chain variable domain having at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, or more changes, e.g., amino acid substitutions or deletions, from the amino acid sequence of any one of AH.1 to AH.85, e.g., AH.1, AH.2, or AH.68, e.g., the amino acid sequence of the FR region in the entire variable region set forth in SEQ ID NO: 10 or SEQ ID NO: 11.

[0101]

[0197] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a light chain framework region 1 of AH.1 or AH.2.

[0102]

[0198] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises light chain framework region 2 of AH.1 or AH.2.

[0103]

[0199] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises the light chain framework region 3 of AH.1 or AH.2.

[0104]

[0200] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises the light chain framework region 4 of AH.1 or AH.2.

[0105]

[0201] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises a light chain variable domain comprising a framework region, e.g., framework region 1 (FR1), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at position 10 according to Kabat numbering. In some embodiments, FR1 comprises a phenylalanine, e.g., a serine-to-phenylalanine substitution, at position 10. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0106]

[0202] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a light chain variable domain comprising a framework region, e.g., framework region 2 (FR2), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at a position described herein according to Kabat numbering. In some embodiments, FR2 comprises a histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a tyrosine to histidine substitution. In some embodiments, FR2 comprises an alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., an arginine to alanine substitution. In some embodiments, the substitution is relative to the human germline light chain framework region sequence.

[0107]

[0203] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a light chain variable domain comprising a framework region, e.g., framework region 3 (FR3), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at a position described herein according to Kabat numbering. In some embodiments, FR3 comprises a phenylalanine at position 87, e.g., a substitution, e.g., a tyrosine to phenylalanine, at position 87 according to Kabat numbering. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

[0108]

[0204] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a light chain variable domain comprising: (a) framework region 1 (FR1) comprising a phenylalanine at position 10, e.g., a substitution at position 10 according to Kabat numbering, e.g., a serine to phenylalanine substitution; (b) framework region 2 (FR2) comprising a histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a tyrosine to histidine substitution, and an alanine at position 46, e.g., an arginine to alanine substitution, e.g., as set forth in the amino acid sequence of SEQ ID NO: 10; and (c) framework region 3 (FR3) comprising a phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a tyrosine to phenylalanine substitution, e.g., as set forth in the amino acid sequence of SEQ ID NO: 10. In some embodiments, the substitution is relative to the human germline light chain framework region sequence.

[0109]

[0205] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a light chain variable domain comprising: (a) framework region 2 (FR2) comprising a histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a tyrosine to histidine substitution, and an alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., an arginine to alanine substitution; and (b) framework region 3 (FR3) comprising a phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a tyrosine to phenylalanine substitution, as set forth in the amino acid sequence of SEQ ID NO: 11. In some embodiments, the substitution is relative to the human germline light chain framework region sequence.

[0110]

[0206] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises a light chain variable domain comprising: (a) framework region 1 (FR1) that contains alterations, e.g., substitutions (e.g., conservative substitutions), at one or more (e.g., all) positions described herein according to Kabat numbering; (b) framework region 2 (FR2) that contains alterations, e.g., substitutions (e.g., conservative substitutions), at one or more (e.g., all) positions described herein according to Kabat numbering; and (c) framework region 3 (FR3) that contains alterations, e.g., substitutions (e.g., conservative substitutions), at one or more (e.g., all) positions described herein according to Kabat numbering. In some embodiments, the substitutions are relative to the human germline light chain framework region sequence.

[0111]

[0207] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * In some embodiments, the anti-TCRβ V antibody molecule comprises the heavy chain framework region 1 of AH.1 or AH.2. In some embodiments, the anti-TCRβ V antibody molecule comprises the heavy chain framework region 1 of AH.1 or AH.2. * 01) antibody molecule comprises heavy chain variable framework region 2 of AH.1 or AH.2. In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises heavy chain variable framework region 3 of AH.1 or AH.2. In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain variable framework region 4 of AH.1 or AH.2.

[0112]

[0208] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises a heavy chain variable domain comprising a framework region, e.g., framework region 3 (FR3), that comprises an alteration, e.g., a substitution (e.g., a conservative substitution), at a position described herein according to Kabat numbering. In some embodiments, FR3 comprises a threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a glutamic acid to threonine substitution. In some embodiments, FR3 comprises a glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., an arginine to glycine substitution. In some embodiments, the substitution is relative to the human germline heavy chain framework region sequence.

[0113]

[0209] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain variable domain comprising framework region 3 (FR3) comprising a threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a glutamic acid to threonine substitution, and a glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., an arginine to glycine substitution, as set forth in the amino acid sequence of SEQ ID NO: 10.

[0114]

[0210] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises heavy chain framework regions 1-4 of AH.1 or AH.2, e.g., SEQ ID NO: 9. In some embodiments, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises light chain framework regions 1-4 of AH.1, e.g., SEQ ID NO: 10. In some embodiments, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises light chain framework regions 1-4 of AH.2, e.g., SEQ ID NO: 11. In some embodiments, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) antibody molecule comprises heavy chain framework regions 1-4 of AH.1, e.g., SEQ ID NO: 9; and light chain framework regions 1-4 of AH.1, e.g., SEQ ID NO: 10. In some embodiments, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5) is used. * 01) The antibody molecule comprises heavy chain framework regions 1-4 of AH.2, e.g., SEQ ID NO: 9; and light chain framework regions 1-4 of AH.2, e.g., SEQ ID NO: 11.

[0115]

[0211] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The heavy or light chain variable domain, or both, of the antibody molecule comprises an amino acid sequence that is substantially identical to an amino acid sequence described herein, e.g., an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to a variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence of Table 1, or an antibody described herein that differs by at least 1 or 5 residues but fewer than 40, 30, 20, or 10 residues from the variable region of an antibody.

[0116]

[0212] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence set forth in Table 1, or a sequence substantially identical thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical, or that differs by no more than 1, 2, 5, 10, or 15 amino acid residues from a sequence set forth in Table 1). In another embodiment, an anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises a VH and / or VL domain encoded by a nucleic acid having a nucleotide sequence set forth in Table 1, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical to a sequence set forth in Table 1, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides).

[0117]

[0213] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a VH domain comprising the amino acid sequence of SEQ ID NO:9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:9 by no more than 1, 2, 5, 10, or 15 amino acid residues; and / or a VL domain comprising the amino acid sequence of SEQ ID NO:10, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:10, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:10 by no more than 1, 2, 5, 10, or 15 amino acid residues.

[0118]

[0214] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a VH domain comprising an amino acid sequence of SEQ ID NO:9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:9 by no more than 1, 2, 5, 10, or 15 amino acid residues; and / or a VL domain comprising an amino acid sequence of SEQ ID NO:11, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO:11, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:11 by no more than 1, 2, 5, 10, or 15 amino acid residues.

[0119]

[0215] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a full-length antibody or a fragment thereof (e.g., Fab, F(ab')2, Fv, single-domain antibody, or single-chain Fv fragment (scFv)). In embodiments, an anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a monoclonal antibody, or an antibody having a single specificity. In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule may also be humanized, chimeric, camelid, shark, or in vitro generated. In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a humanized antibody molecule. An anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The heavy and light chains of the antibody molecule may be full length (e.g., the antibody may comprise at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or may comprise an antigen-binding fragment (e.g., a Fab, F(ab'), Fv, a single-chain Fv fragment, a single-domain antibody, a diabody (dAb), a bivalent antibody, or a bispecific antibody or fragment thereof, a single-domain variant thereof, or a camelid antibody).

[0120]

[0216] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a multifunctional molecule, e.g., a form of bispecific molecule as described herein.

[0121]

[0217] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule has a heavy chain constant region (Fc) selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is selected from the heavy chain constant region of IgG1 or IgG2 (e.g., human IgG1 or IgG2). In some embodiments, the heavy chain constant region is human IgG1. In some embodiments, the Fc region comprises an Fc region variant, e.g., as described herein.

[0122]

[0218] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule has a light chain constant region selected from the light chain constant region of kappa or lambda, preferably kappa (e.g., human kappa). In some embodiments, the constant region is selected from the light chain constant region of an anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule has been altered, e.g., mutated, to modify its properties (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). For example, the constant region is mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding, e.g., compared to human IgG1 (e.g., the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K), and 314 (N to F) of SEQ ID NO: 212 or 214; or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K), and 317 (N to F) of SEQ ID NO: 215, 216, 217, or 218).

[0123]

[0219] Antibody AH.1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 72. Antibody AH.2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 3279. Antibody AH.68 comprises the amino acid sequence of SEQ ID NO: 1337, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto. Antibody AH.69 comprises the amino acid sequence of SEQ ID NO: 1500, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto.

[0124]

[0220] Additional exemplary humanized anti-TCRβ V6 antibodies are provided in Table 1. In some embodiments, the anti-TCRβ V6 is Antibody A, e.g., humanized Antibody A (Antibody AH), provided in Table 1. In some embodiments, the anti-TCRβ V antibody comprises one or more (e.g., all three) of the LC CDR1, LC CDR2, and LC CDR3 provided in Table 1, and / or one or more (e.g., all three) of the HC CDR1, HC CDR2, and HC CDR3 provided in Table 1, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto. In some embodiments, Antibody A comprises a variable heavy chain (VH) and / or variable light chain (VL) provided in Table 1, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity thereto.

[0125]

[0221] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) Antibody molecules are AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH.7, AH.8, AH.9, AH.10, AH.11, AH.12, AH.13, AH.14, AH.15, AH.16, AH.17, AH.18, AH.19, AH.20, AH.21, AH.22, AH.23, AH.24, A H.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH.36, A H.37, AH.38, AH.39, AH.40, AH.1, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH. 49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH. 61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.7 and a VH of AH.3, AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, or AH.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0126]

[0222] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) Antibody molecules are AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH.7, AH.8, AH.9, AH.10, AH.11, AH.12, AH.13, AH.14, AH.15, AH.16, AH.17, AH.18, AH.19, AH.20, AH.21, AH.22, AH.23, AH.24, A H.25, AH.26, AH.27, AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH.36, A H.37, AH.38, AH.39, AH.40, AH.1, AH.42, AH.43, AH.44, AH.45, AH.46, AH.47, AH.48, AH. 49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH. 61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.7 and a VL of AH.3, AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, or AH.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0127]

[0223] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01)Different types are AH.1, AH.2, AH.3, AH.4, AH.5, AH.6, AH.7, AH.8, AH.9 AH.10, AH.11, AH.12, AH.13, AH.14, AH.15, AH.16, AH.17, AH AH.19, AH.20, AH.21, AH.22, AH.23, AH.24, AH.25, AH.26, AH AH.28, AH.29, AH.30, AH.31, AH.32, AH.33, AH.34, AH.35, AH AH.37, AH.38, AH.39, AH.40, AH.1, AH.42, AH.43, AH.44, AH.45 AH.46, AH.47, AH.48, AH.49, AH.50, AH.51, AH.52, AH.53, AH.54 AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH.61, AH.62, AH.63 AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72 AH.73, AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH AH.82, AH.83, AH.84, AH.85, 80%, 85%, 90%, 95 %, 96%, 97%, 98%, and 99% of the respective ranges of VH; H.3, AH.4, AH.5, AH.6, AH.7, AH.8, AH.9, AH.10, AH.11, AH.12, AH .13、AH.14、AH.15、AH.16、AH.17、AH.18、AH.19、AH.20、AH.21、AH .22、AH.23、AH.24、AH.25、AH.26、AH.27、AH.28、AH.29、AH.30、AH .31、AH.32、AH.33、AH.34、AH.35、AH.36、AH.37、AH.38、AH.39、AH .40、AH.1、AH.42、AH.43、AH.44、AH.45、AH.46、AH.47、AH.48、AH.49, AH.50, AH.51, AH.52, AH.53, AH.54, AH.55, AH.56, AH.57, AH.58, AH.59, AH.60, AH. 61, AH.62, AH.63, AH.64, AH.65, AH.66, AH.67, AH.68, AH.69, AH.70, AH.71, AH.72, AH.7 and AH.3, AH.74, AH.75, AH.76, AH.77, AH.78, AH.79, AH.80, AH.81, AH.82, AH.83, AH.84, or AH.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0128]

[0224] Exemplary anti-TCRβV antibody molecules and the corresponding TCRβV subfamilies recognized by said anti-TCRβV antibody molecules are disclosed in Table 10A.

[0225] Various TCRβV subfamilies and / or subfamily members can be expressed at varying levels in individuals, e.g., healthy individuals, as disclosed in Kitaura K. et al. (2016), BMC Immunology, vol. 17:38, the entire contents of which are hereby incorporated by reference. For example, TCRβV6-5 is expressed in approximately 3-6% of healthy donors.

[0129]

[0226] The expression of various TCRβ subfamilies and / or subfamily members may also vary in cancer cells. For example, TCRβV is present in approximately 3-6% of tumor-infiltrating T cells, regardless of tumor type (see Li B. et al., Nature Genetics, 2016, vol:48(7):725-32, the entire contents of which are hereby incorporated by reference). Li et al. also disclose that TCRβV6-5 is frequently present in tumor cells.

[0130] Anti-TCRβ V6 antibody

[0227] In one aspect, as used herein, human TCRβ V6, e.g., TCRβ V6-4 * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5 * 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01, or TCRβ V6-1 * In some embodiments, the TCRβ V6 subfamily comprises TCRβ V6-5. * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-4 * 02 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-9 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-8 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-5 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 02 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-6 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-2 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-3 * 01 or a variant thereof. In some embodiments, the TCRβ V6 comprises TCRβ V6-1 * 01 or its variants.

[0131]

[0228] In some embodiments, TCRβ V6-5 * 01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or more identity thereto. * 01 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having 85%, 90%, 95%, 99% or more identity thereto.

[0132]

[0229] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a non-murine antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the antibody molecule is an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a human antibody molecule. In some embodiments, the antibody molecule is an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is a humanized antibody molecule.

[0133]

[0230] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule is isolated or recombinant.

[0134]

[0231] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one antigen-binding region, e.g., a variable region or antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0135]

[0232] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, three, or four variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0136]

[0233] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody molecule described in Table 1, or an antibody encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0137]

[0234] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by a nucleotide sequence in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0138]

[0235] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises a heavy chain constant region of IgG4, e.g., human IgG4. In yet another embodiment, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a heavy chain constant region of IgG1, e.g., human IgG1. In some embodiments, the heavy chain constant region comprises an amino acid sequence set forth in Table 3, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0139]

[0236] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a kappa light chain constant region, e.g., a human kappa light chain constant region. In some embodiments, the light chain constant region comprises an amino acid sequence set forth in Table 3, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0140]

[0237] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region (VH) of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence of Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0141]

[0238] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising the amino acid sequence shown in Table 1, or the amino acid sequence encoded by the nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1 or the amino acid sequence encoded by the nucleotide sequence shown in Table 1.

[0142]

[0239] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three complementarity determining regions (CDRs) from the light chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1 through AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence of Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0143]

[0240] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence set forth in Table 1, or an amino acid sequence encoded by a nucleotide sequence set forth in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence set forth in Table 1, or the amino acid sequence encoded by the nucleotide sequence set forth in Table 1.

[0144]

[0241] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, three, four, five, or six CDRs (or collectively all of the CDRs) from heavy and light chain variable regions comprising the amino acid sequence shown in Table 1, or the amino acid sequence encoded by the nucleotide sequence shown in Table 1. In some embodiments, one or more of the CDRs (or collectively all of the CDRs) comprise one, two, three, four, five, six, or more changes, e.g., amino acid substitutions or deletions, compared to the amino acid sequence shown in Table 1 or the amino acid sequence encoded by the nucleotide sequence shown in Table 1.

[0145]

[0242] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises all six CDRs from an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by a nucleotide sequence in Table 1, or closely related CDRs, e.g., CDRs that are identical or have at least one amino acid change, but have no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In some embodiments, an anti-TCRβ V antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule may comprise any of the CDRs described herein.

[0146]

[0243] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to Kabat's definition as set forth in Table 1) from the heavy chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid alteration but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Kabat et al. as set forth in Table 1.

[0147]

[0244] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to Kabat's definition as set forth in Table 1) from the light chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid alteration but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Kabat et al. as set forth in Table 1.

[0148]

[0245] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat definition set forth in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody selected from any one of AH.1 through AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequences of Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid alteration but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, three, four, five, or six CDRs according to Kabat shown in Table 1.

[0149]

[0246] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises all six CDRs according to Kabat et al. (e.g., all six CDRs according to Kabat's definition as set forth in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence of Table 1; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to all six CDRs according to Kabat et al. as set forth in Table 1. In some embodiments, an anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule may comprise any of the CDRs described herein.

[0150]

[0247] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three hypervariable loops having the same canonical structure as the corresponding hypervariable loops of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, e.g., the same canonical structure as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domain of an antibody described herein. See, e.g., Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798, for a description of the canonical structure of hypervariable loops. These structures can be determined by inspection of the tables set forth in these references.

[0151]

[0248] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition in Table 1) from the heavy chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1 through AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid alteration but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Chothia et al. shown in Table 1.

[0152]

[0249] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition in Table 1) from the light chain variable region of an antibody described herein, e.g., an antibody selected from any one of AH.1 through AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid alteration but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, or three CDRs according to Chothia et al. shown in Table 1.

[0153]

[0250] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises at least one, two, three, four, five, or six CDRs according to Chothia et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Chothia definition as set forth in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody selected from any one of AH.1 through AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequences of Table 1; or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence that has at least one amino acid alteration but no more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to one, two, three, four, five, or six CDRs according to Chothia as set forth in Table 1.

[0154]

[0251] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecule comprises all six CDRs according to Chothia et al. (e.g., all six CDRs according to the Chothia definition as set forth in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody selected from any one of AH.1-AH.85, e.g., AH.1, AH.2, or AH.68, or an antibody described in Table 1, or an antibody encoded by the nucleotides of Table 1; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences; or a sequence having at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) compared to all six CDRs according to Chothia et al. as set forth in Table 1. In some embodiments, an anti-TCRβ antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule may comprise any of the CDRs described herein.

[0155]

[0252] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises a combination of CDRs or hypervariable loops as defined according to Kabat et al., Chothia et al., or as set forth in Table 1.

[0156]

[0253] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) An antibody molecule may contain any combination of CDRs or hypervariable loops according to the definition of Kabat and Chothia.

[0157]

[0254] In some embodiments, the CDR combinations listed in Table 1 are CDRs that include Kabat CDRs and Chothia CDRs.

[0255] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) antibody molecules comprise a combination of CDRs or hypervariable loops identified as the combined CDRs in Table 1. In some embodiments, an anti-TCRβ V antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule may contain any combination of CDRs or hypervariable loops according to the "combined" CDRs set forth in Table 1.

[0158]

[0256] In some embodiments, e.g., embodiments comprising variable regions, CDRs (e.g., combined CDRs, Chothia CDRs, or Kabat CDRs), or other sequences referred to herein, e.g., in Table 1, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic antibody molecule, or an antibody molecule comprising an antigen-binding fragment of an antibody, e.g., a half antibody or an antigen-binding fragment of a half antibody. In certain embodiments, the antibody molecule comprises a multifunctional molecule, e.g., a bispecific molecule, e.g., as described herein.

[0159]

[0257] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises (i) one, two, or all of light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO:2, SEQ ID NO:10, or SEQ ID NO:11, and / or (ii) one, two, or all of heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO:1 or SEQ ID NO:9.

[0160]

[0258] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO:2, and HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO:1.

[0161]

[0259] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 10, and HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0162]

[0260] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 11, and HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

[0163]

[0261] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises (i) the LC CDR1 amino acid sequence of SEQ ID NO: 6, the LC CDR2 amino acid sequence of SEQ ID NO: 7, or the LC CDR3 amino acid sequence of SEQ ID NO: 8; and / or (ii) the HC CDR1 amino acid sequence of SEQ ID NO: 3, the HC CDR2 amino acid sequence of SEQ ID NO: 4, or the HC CDR3 amino acid sequence of SEQ ID NO: 5.

[0164]

[0262] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises (i) a light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 6, the LC CDR2 amino acid sequence of SEQ ID NO: 7, or the LC CDR3 amino acid sequence of SEQ ID NO: 8; and / or (ii) a heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 3, the HC CDR2 amino acid sequence of SEQ ID NO: 4, or the HC CDR3 amino acid sequence of SEQ ID NO: 5.

[0165]

[0263] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises (i) the LC CDR1 amino acid sequence of SEQ ID NO: 51, the LC CDR2 amino acid sequence of SEQ ID NO: 52, or the LC CDR3 amino acid sequence of SEQ ID NO: 53; and / or (ii) the HC CDR1 amino acid sequence of SEQ ID NO: 45, the HC CDR2 amino acid sequence of SEQ ID NO: 46, or the HC CDR3 amino acid sequence of SEQ ID NO: 47.

[0166]

[0264] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises (i) a light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 51, the LC CDR2 amino acid sequence of SEQ ID NO: 52, or the LC CDR3 amino acid sequence of SEQ ID NO: 53; and / or (ii) a heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 45, the HC CDR2 amino acid sequence of SEQ ID NO: 46, or the HC CDR3 amino acid sequence of SEQ ID NO: 47.

[0167]

[0265] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 *01) The antibody molecule comprises (i) the LC CDR1 amino acid sequence of SEQ ID NO: 54, the LC CDR2 amino acid sequence of SEQ ID NO: 55, or the LC CDR3 amino acid sequence of SEQ ID NO: 56; and / or (ii) the HC CDR1 amino acid sequence of SEQ ID NO: 48, the HC CDR2 amino acid sequence of SEQ ID NO: 49, or the HC CDR3 amino acid sequence of SEQ ID NO: 50.

[0168]

[0266] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises (i) a light chain variable region (VL) comprising the LC CDR1 amino acid sequence of SEQ ID NO: 54, the LC CDR2 amino acid sequence of SEQ ID NO: 55, or the LC CDR3 amino acid sequence of SEQ ID NO: 56; and / or (ii) a heavy chain variable region (VH) comprising the HC CDR1 amino acid sequence of SEQ ID NO: 48, the HC CDR2 amino acid sequence of SEQ ID NO: 49, or the HC CDR3 amino acid sequence of SEQ ID NO: 50.

[0169]

[0267] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises the VH and / or VL of an antibody described in Table 1, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0170]

[0268] In some embodiments, an anti-TCRβ antibody molecule, such as anti-TCRβ V6 (e.g., anti-TCRβ V6-5 * 01) The antibody molecule comprises the VH and VL of an antibody described in Table 1, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0171]

[0269] In some embodiments, the anti-TCRVb antibodies described herein have an antigen-binding domain having a VL with the consensus sequence of SEQ ID NO: 230, wherein position 30 is G, E, A, or D; position 31 is N or D; position 32 is R or K; position 36 is Y or H; and / or position 56 is K or S.

[0172]

[0270] In some embodiments, the anti-TCRVb antibodies described herein have an antigen-binding domain having a VH with the consensus sequence of SEQ ID NO: 231, wherein position 27 is H, T, G, or Y; position 28 is D, T, or S; position 30 is H, R, D, K, or T; position 31 is L, D, K, T, or N; position 32 is W, F, T, I, Y, or G; position 49 is R or W; position 50 is V, I, or F; position 51 is F, S, or Y; position 52 is A or P; position 56 is N or S; position 57 is T, V, Y, or I; position 58 is K or R; position 97 is G or V; position 99 is Y or I; position 102 is Y or A; and / or position 103 is D or G.

[0173] Anti-TCRβ V10 antibody

[0271] In one aspect, provided herein is an anti-TCRβV antibody molecule that binds to a human TCRβ V10 subfamily member. In some embodiments, the TCRβ V10 subfamily is also known as TCRβ V12. In some embodiments, the TCRβ V10 subfamily is TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01 or TCRβ V10-2 * 01, or any variant thereof.

[0174]

[0272] Exemplary anti-TCR β V10 antibodies are provided in Table 12. In some embodiments, the anti-TCR β V10 is antibody D, e.g., humanized antibody D (antibody DH) provided in Table 12. In some embodiments, antibody D comprises one or more (e.g., three) light chain CDRs and / or one or more (e.g., three) heavy chain CDRs provided in Table 12, or a sequence with at least 95% sequence identity thereto. In some embodiments, antibody D comprises a variable heavy chain (VH) and / or a variable light chain (VL) provided in Table 12, or a sequence with at least 95% sequence identity thereto.

[0175]

[0273] In some embodiments, the anti-TCR β V10 antibody molecule comprises the VH or VL of an antibody described in Table 12, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0176]

[0274] In some embodiments, the anti-TCR β V10 antibody molecule comprises the VH and VL of an antibody set forth in Table 12, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0177] Antibody-like frameworks or scaffolds

[0275] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be used in the anti-TCRvb antibody molecules or multifunctional formats described herein, as long as the resulting polypeptide contains at least one binding region that specifically binds to a target antigen, such as a TCRvb, tumor antigen, among others. Such frameworks or scaffolds include the five major idiotypes of human immunoglobulins or fragments thereof, and preferably include immunoglobulins of other animal species with humanized aspects. New frameworks, scaffolds, and fragments are constantly being discovered and developed by those skilled in the art.

[0178]

[0276] In some embodiments, the anti-TCRvb antibody molecules or multifunctional formats thereof described herein include non-immunoglobulin-based antibodies that use non-immunoglobulin scaffolds onto which CDRs can be grafted. Any non-immunoglobulin framework and scaffold can be used as long as it contains a binding region specific for the target antigen (e.g., TCRvb or tumor antigen). Exemplary non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden), and affilin (gamma-cristatin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0179]

[0277] Fibronectin scaffolds are typically based on fibronectin type III domains (e.g., the 10th fibronectin type III module (10 Fn3 domain)). Fibronectin type III domains have seven or eight beta strands distributed between two beta strands, which compress against each other to form the core of the protein and further contain solvent-exposed loops (similar to CDRs) connecting the beta strands to each other. At least three such loops are present at each end of the beta strand sandwich, and the ends are at the protein interface perpendicular to the beta sheet direction (see U.S. Pat. No. 6,818,418). Due to this structure, non-immunoglobulin antibodies essentially mimic the antigen-binding properties and affinity of antibodies. These scaffolds can be used for in vitro loop randomization and shuffling strategies similar to the in vivo antibody affinity maturation process. These fibronectin-based molecules can be used as scaffolds to exchange the loop regions of the molecule for the CDRs of the present invention using standard cloning techniques.

[0180]

[0278] Ankyrin technology is based on the use of proteins with ankyrin-derived repeat modules as scaffolds to carry variable regions that can be used to bind to different targets. Ankyrin repeat modules are typically polypeptides of approximately 33 amino acids consisting of two antiparallel α-helices and a β-turn. The binding of the variable regions can be optimized using ribosome display.

[0181]

[0279] Avimers are primarily used for protein-protein interactions, and over 250 human proteins are structurally based on A domains. Avimers consist of multiple distinct "A domain" monomers (2-10) linked via amino acid linkers. For example, avimers capable of binding to target antigens can be generated using the methodologies described in U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

[0182]

[0280] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on a scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein of the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (see, e.g., U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies; they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. Despite their small size, the binding site of affibody molecules is similar to that of antibodies.

[0183]

[0281] Anticalins are known and commercially available, for example, from Pieris ProteoLab AG. They are derived from lipocalins, a broad group of small, robust proteins typically involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins are present in human tissues and body fluids. The protein structure is reminiscent of immunoglobulins, with hypervariable loops located on top of a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain with 160–180 amino acid residues, slightly larger than a single immunoglobulin domain. The set of four loops that constitute the binding pocket exhibits remarkable structural plasticity and tolerates diverse side chains. Thus, the binding site can be reshaped in a unique process to recognize defined target molecules of different shapes with high affinity and specificity. One protein in the lipocalin family, bilin-binding protein (BBP) from Pieris brassicae, has been used to develop anticalins by mutagenizing the set of four loops. An example of a patent application describing anticalins is PCT application WO 199916873.

[0184]

[0282] Affilin molecules are small, non-immunoglobulin proteins designed for specific affinity to proteins and small molecules. Novel affilin molecules can be rapidly selected from two libraries, each based on a different human-derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are used: one is gamma-crystallin, a structural protein in the human eye lens, and the other is a "ubiquitin" superfamily protein. Both human scaffolds are very small, stable at high temperatures, and largely resistant to pH changes and denaturing agents. This high stability is primarily due to the protein's extended beta-sheet structure. Examples of gamma-crystallin-derived proteins are described in International Publication No. 200104144, and examples of "ubiquitin-like" proteins are described in International Publication No. 2004106368.

[0185]

[0283] Protein epitope mimetics (PEMs) are moderately sized cyclic peptide-like molecules (molecular weight 1-2 kDa) that mimic the beta-hairpin secondary structure of proteins, which is the major secondary structure involved in protein-protein interactions.

[0186]

[0284] Domain antibodies (dAbs) may be used in the anti-TCRvb antibody molecules described herein, or their multifunctional formats, are small functional binding fragments of antibodies corresponding to the variable regions of either the heavy or light chains of the antibody. Domain antibodies have been successfully expressed in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, e.g., U.S. Patent Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patent Nos. 0368684 & 0616640; WO 05 / 035572, WO 04 / 101790, WO 04 / 081026, WO 04 / 058821, WO 04 / 003019, and WO 03 / 002609). Nanobodies are derived from the heavy chain of an antibody.

[0187]

[0285] Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Patent No. 6,765,087, U.S. Patent No. 6,838,254, and International Publication No. WO 06 / 079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies can be created by removing the hinge region of an IgG4 antibody. Further details of unibodies and methods for preparing them can be found in International Publication No. WO 2007 / 059782.

[0188] Anti-TCRVβ antibody effector functions and Fc variants

[0286] In some embodiments, the anti-TCRVβ antibodies described herein comprise an Fc region, e.g., as described herein. In some embodiments, the Fc region is a wild-type Fc region, e.g., a wild-type human Fc region. In some embodiments, the Fc region comprises a variant, e.g., an Fc region that includes an addition, substitution, or deletion of at least one amino acid residue that results in the Fc region having, e.g., reduced or eliminated affinity for, at least one Fc receptor.

[0189]

[0287] The Fc region of an antibody interacts with multiple receptors or ligands, including Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIIA), complement protein CIq, and other molecules such as proteins A and G. These interactions are essential for diverse effector functions and downstream signaling events, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0190]

[0288] In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has reduced, e.g., eliminated, affinity for an Fc receptor, e.g., an Fc receptor described herein, in some embodiments, the reduced affinity is compared to an otherwise similar antibody with the wild-type Fc region.

[0191]

[0289] In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP, and / or CDC); (2) reduced binding to one or more Fc receptors; and / or (3) reduced binding to C1q complement. In some embodiments, the reduction in any one or all of properties (1)-(3) is relative to a wild-type Fc region and an otherwise similar antibody.

[0192]

[0290] In some embodiments, anti-TCRVβ antibodies comprising a variant Fc region have reduced affinity for a human Fc receptor, e.g., FcγRI, FcγRII, and / or FcγRII. In some embodiments, anti-TCRVβ antibodies comprising a variant Fc region comprise a human IgG1 region or a human IgG4 region.

[0193]

[0291] In some embodiments, anti-TCR Vβ antibodies comprising a variant Fc region activate and / or expand T cells, e.g., as described herein. In some embodiments, anti-TCR Vβ antibodies comprising a variant Fc region have a cytokine profile that differs from the cytokine profile described herein, e.g., the cytokine profile of a T cell engager that binds to a receptor or molecule other than the TCR β V region (a "non-TCR β V-binding T cell engager"). In some embodiments, a non-TCR β V-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., a CD3 epsilon (CD3e) molecule); or a TCR alpha (TCR α) molecule.

[0194]

[0292] Exemplary Fc region variants are provided in Table 14 and are also disclosed in Saunders O, (2019) Frontiers in Immunology; vol 10, article 1296, the entire contents of which are hereby incorporated by reference.

[0195]

[0293] In some embodiments, the anti-TCRVβ antibodies described herein comprise any one or all, or any combination, of the Fc regions disclosed in Table 14.

[0294] In some embodiments, the anti-TCRVβ antibodies described herein comprise any one or all, or any combination of, Fc region variants, e.g., mutations, disclosed in Table 14. In some embodiments, the anti-TCRVβ antibodies described herein comprise an Asn297Ala (N297A) mutation. In some embodiments, the anti-TCRVβ antibodies described herein comprise Leu234Ala / Leu235Ala (LALA) mutations.

[0196] multifunctional molecules

[0295] The terms "multifunctional molecule" and "multispecific molecule" are used interchangeably herein and refer to a molecule, e.g., a polypeptide, having two or more functionalities, e.g., two or more binding specificities. In some embodiments, the functionalities may include one or more immune cell engagers, one or more tumor-binding molecules, one or more cytokine molecules, one or more stromal modifiers, and other moieties described herein. In some embodiments, the multifunctional molecule is a multifunctional antibody molecule, e.g., a bispecific antibody molecule. In some embodiments, the multifunctional molecule comprises an anti-TCRVb antibody molecule described herein.

[0197]

[0296] Provided herein, in certain embodiments, is a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and at least one cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first portion of a first T cell receptor variable beta (TCRβV) binding portion and a first dimerization module linked to the first portion of the first TCRβV binding portion; (ii) the second polypeptide comprises a first portion of a first T cell receptor variable beta (TCRβV) binding portion and a first dimerization module linked to the first portion of the first TCRβV binding portion; and (iv) a fourth polypeptide comprises the second portion of the second TCRβV binding portion; and at least one cytokine polypeptide, or a functional fragment or functional variant thereof, is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof.

[0198]

[0297]

[0013] In certain embodiments, the present specification describes a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and at least one cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first portion of a first TCRβV binding portion and a first dimerization module linked to the first portion of the first TCRβV binding portion; (ii) the second polypeptide comprises a second portion of the first TCRβV binding portion; and (iii) the third polypeptide comprises a second dimerization module; and the at least one cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, or a combination thereof.

[0199]

[0298]

[0013] In certain embodiments, the present specification describes a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and at least one cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous, and (i) the first polypeptide comprises a first portion of a first TCRβV binding moiety and a first dimerization module linked to the first portion of the first TCRβV binding moiety; (ii) the second polypeptide comprises a second portion of the first TCRβV binding moiety; and (iii) the third polypeptide comprises a second dimerization module; and the at least one cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, the third polypeptide, or a combination thereof; and the multifunctional polypeptide molecule does not comprise any additional TCRβV binding moieties other than the first TCRβV binding moiety.

[0200]

[0299] In some embodiments, the first portion of the first TCRβV-binding moiety comprises a first heavy chain variable domain (VH) and a first heavy chain constant domain 1 (CH1) linked to the first VH. In some embodiments, the first CH1 is linked to the C-terminus of the first VH. In some embodiments, the second portion of the first TCRβV-binding moiety comprises a first light chain variable domain (VL) and a first light chain constant domain (CL) linked to the first VL. In some embodiments, the first CL is linked to the C-terminus of the first VL. In some embodiments, a first dimerization module is linked to the first portion of the first TCRβV-binding moiety. In some embodiments, the first dimerization module is linked to the C-terminus of the first portion of the first TCRβV-binding moiety. In some embodiments, the first portion of the second TCRβV binding moiety comprises a second VH and a second CH1 linked to the second VH. In some embodiments, the second CH1 is linked to the C-terminus of the second VH. In some embodiments, the second portion of the second TCRβV binding moiety comprises a second VL and a second CL linked to the second VL. In some embodiments, the second CL is linked to the C-terminus of the second VL. In some embodiments, a second dimerization module is linked to the first portion of the second TCRβV binding moiety. In some embodiments, the second dimerization module is linked to the C-terminus of the first portion of the second TCRβV binding moiety.

[0201]

[0300] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (c a) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide, or a functional fragment or functional variant thereof; and a C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (d) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide, or a functional fragment or functional variant thereof; and a C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; or (e) a combination thereof.

[0202]

[0301] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; and (a-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; and (b-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide, or a functional fragment or functional variant thereof. (c-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; and (c-2) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; (d-1) the N-terminus of the second polypeptide is linked to the third cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to the fourth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof;and (d-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; (e-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; and (e-2) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a functional fragment or functional variant thereof; the fourth or (f-1) the N-terminus of the third polypeptide is linked to the fifth cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to the sixth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; and (f-2) the N-terminus of the fourth polypeptide is linked to the seventh cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the fourth polypeptide is linked to the eighth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof;

[0203]

[0302] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (a-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; and (a-3) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide, or a functional fragment or functional variant thereof; (b-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; (b-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; and (b-3) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof;(c-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; and (c-3) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide or a functional fragment or functional variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof;

[0204]

[0303] In some embodiments, (1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; and the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof. (3) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; and (4) the N-terminus of the fourth polypeptide is linked to a seventh cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof.

[0205]

[0304] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the second polypeptide, the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the third polypeptide, and the seventh cytokine polypeptide, the eighth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the fourth polypeptide, or a combination thereof.

[0206]

[0305] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (c) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; or (d) a combination thereof.

[0207]

[0306] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; and (a-2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to the first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to the second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof. and (b-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a functional fragment or functional variant thereof; and the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; or (c-1) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide or a functional fragment or functional variant thereof; and the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof; and (c-2) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide or a functional fragment or functional variant thereof; and the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or functional variant thereof; or a combination thereof.

[0208]

[0307] In some embodiments, (1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (2) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; and (3) the N-terminus of the third polypeptide is linked to a fifth cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof.

[0209]

[0308] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the second polypeptide, and the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the third polypeptide, or a combination thereof.

[0210]

[0309] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRβV-binding moiety and the first dimerization module, a linker between the first portion of the second TCRβV-binding moiety and the second dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between the second VH and the second CH1, a linker between the second VL and the second CL, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the first polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the second polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and a third polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and a fourth polypeptide, or a combination thereof.

[0211]

[0310] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRβV-binding portion and the first dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the first polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the second polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the third polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is a peptide linker, and the linker is a GS linker. In some embodiments, the linker is a peptide linker, and the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0212]

[0311] Provided herein, in certain embodiments, is a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a first cytokine polypeptide or a functional fragment or functional variant thereof, and a second cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first portion of a first TCRβV-binding moiety and a first dimerization module linked to the first portion of the first TCRβV-binding moiety; (ii) the second polypeptide (iii) a third polypeptide comprises a first portion of a second TCRβV-binding portion and a second dimerization module linked to the first portion of the second TCRβV-binding portion; and (iv) a fourth polypeptide comprises a second portion of a second TCRβV-binding portion; and a first cytokine polypeptide, or a functional fragment or functional variant thereof, is covalently linked to the C-terminus of the second polypeptide, and a second cytokine polypeptide, or a functional fragment or functional variant thereof, is covalently linked to the C-terminus of the fourth polypeptide.

[0213]

[0312] Provided herein, in certain embodiments, is a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a cytokine polypeptide, or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first portion of a first TCRβV-binding moiety and a first dimerization module linked to the first portion of the first TCRβV-binding moiety; (ii) the second polypeptide comprises a first portion of a first TCRβV-binding moiety and a first dimerization module linked to the first portion of the first TCRβV-binding moiety; (iii) a third polypeptide comprises a first portion of a second TCRβV-binding portion and a second dimerization module linked to the first portion of the second TCRβV-binding portion; and (iv) a fourth polypeptide comprises a second portion of the second TCRβV-binding portion; and a cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the C-terminus of the second polypeptide or the C-terminus of the fourth polypeptide.

[0214]

[0313] Provided herein, in certain embodiments, is a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a cytokine polypeptide, or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first portion of a first TCRβV-binding moiety and a first dimerization module linked to the first portion of the first TCRβV-binding moiety; (ii) the second polypeptide comprises a first portion of a first TCRβV-binding moiety and a first dimerization module linked to the first portion of the first TCRβV-binding moiety; (iii) a third polypeptide comprises a first portion of a second TCRβV-binding portion and a second dimerization module linked to the first portion of the second TCRβV-binding portion; and (iv) a fourth polypeptide comprises a second portion of the second TCRβV-binding portion; and a cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the C-terminus of the first polypeptide or the C-terminus of the third polypeptide.

[0215]

[0314]

[0013] In certain embodiments, the present specification describes a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and a cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous, and (i) the first polypeptide comprises a first portion of a first TCRβV binding portion and a first dimerization module linked to the first portion of the first TCRβV binding portion; (ii) the second polypeptide comprises a second portion of the first TCRβV binding portion; and (iii) the third polypeptide comprises a second dimerization module; at least one cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the N-terminus of the third polypeptide; and the multifunctional polypeptide molecule does not comprise any additional TCRβV binding portions other than the first TCRβV binding portion.

[0216]

[0315] In some embodiments, the first portion of the first TCRβV-binding portion comprises a first VH and a first CH1 linked to the first VH. In some embodiments, the first CH1 is linked to the C-terminus of the first VH.

[0217]

[0316] In some embodiments, the second portion of the first TCRβV-binding portion comprises a first VL and a first CL linked to the first VL, hi some embodiments, the first CL is linked to the C-terminus of the first VL.

[0218]

[0317] In some embodiments, the first dimerization module is linked to the first portion of the first TCRβV binding moiety. In some embodiments, the first dimerization module is linked to the C-terminus of the first portion of the first TCRβV binding moiety. In some embodiments, the first portion of the second TCRβV binding moiety comprises a second VH and a second CH1 linked to the second VH. In some embodiments, the second CH1 is linked to the C-terminus of the second VH. In some embodiments, the second portion of the second TCRβV binding moiety comprises a second VL and a second CL linked to the second VL. In some embodiments, the second CL is linked to the C-terminus of the second VL. In some embodiments, the second dimerization module is linked to the first portion of the second TCRβV binding moiety. In some embodiments, the second dimerization module is linked to the C-terminus of the first portion of the second TCRβV binding moiety.

[0219]

[0318] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRβV-binding moiety and the first dimerization module, a linker between the first portion of the second TCRβV-binding moiety and the second dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between the second VH and the second CH1, a linker between the second VL and the second CL, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the first polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the second polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and a third polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and a fourth polypeptide, or a combination thereof. In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first portion of the first TCRβV-binding portion and the first dimerization module, a linker between the first VH and the first CH1, a linker between the first VL and the first CL, a linker between at least one cytokine polypeptide, or a functional fragment or variant thereof, and a third polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is a peptide linker, and the linker is a GS linker. In some embodiments, the linker is a peptide linker, and the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0220]

[0319] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises any one selected from the group consisting of a Fab, a F(ab')2, an Fv, a single-chain Fv (scFv), a single-domain antibody, a diabody (dAb), a camelid antibody, and a combination thereof. In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises an scFv or a Fab.

[0221]

[0320] In some embodiments, the multifunctional polypeptide molecule does not comprise an additional antigen binding moiety other than the TCRβV binding moiety, hi some embodiments, the multifunctional polypeptide molecule further comprises an additional antigen binding moiety that is not a TCRβV binding moiety.

[0222]

[0321]

[0013] As used herein, in certain embodiments, there is provided a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide and the second polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first TCRβV-binding moiety and a first dimerization module linked to the C-terminus of the first TCRβV-binding moiety, the first TCRβV-binding moiety comprising a first VL and a first VH; and (ii) the second polypeptide comprises a second A multifunctional polypeptide molecule is described, which comprises a TCRβV binding portion and a second dimerization module linked to the C-terminus of the second TCRβV binding portion; at least one cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, or a combination thereof; the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises an scFv; and the multifunctional polypeptide molecule does not comprise any additional antigen-binding portion other than the first TCRβV binding portion and the second TCRβV binding portion.

[0223]

[0322] Provided herein, in certain embodiments, is a multifunctional polypeptide molecule comprising a first polypeptide, a second polypeptide, and at least one cytokine polypeptide or a functional fragment or functional variant thereof, wherein the first polypeptide and the second polypeptide are discontinuous, and wherein (i) the first polypeptide comprises a first TCRβV-binding moiety and a first dimerization module linked to the C-terminus of the first TCRβV-binding moiety, the first TCRβV-binding moiety comprising a first VL and a first VH; and (ii) a second and wherein the polypeptide of the present invention comprises a second dimerization module; at least one cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the first polypeptide, the second polypeptide, or a combination thereof; the first TCRβV binding portion comprises an scFv; the multifunctional polypeptide molecule does not comprise any additional antigen binding portion other than the first TCRβV binding portion; and wherein the multifunctional polypeptide molecule does not comprise any additional TCRβV binding portion other than the first TCRβV binding portion.

[0224]

[0323] In some embodiments, (a) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; (b) the N-terminus of the second polypeptide is linked to a third cytokine polypeptide, or a functional fragment or functional variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide, or a functional fragment or functional variant thereof; or a combination thereof; or (e) a combination thereof.

[0225]

[0324] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the first polypeptide, and the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is present in a single contiguous polypeptide chain of the second polypeptide, or a combination thereof.

[0226]

[0325] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first TCRβV binding moiety and the first dimerization module, a linker between the second TCRβV binding moiety and the second dimerization module, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the first polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or functional variant thereof, and the second polypeptide, or a combination thereof.

[0227]

[0326] In some embodiments, the multifunctional polypeptide molecule described herein further comprises a linker between the first TCRβV-binding portion and the first dimerization module, a linker between at least one cytokine polypeptide, or a functional fragment or variant thereof, and the first polypeptide, a linker between at least one cytokine polypeptide, or a functional fragment or variant thereof, and the second polypeptide, or a combination thereof. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker is a peptide linker, and the linker is a GS linker. In some embodiments, the linker is a peptide linker, and the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0228]

[0327] In some embodiments, a multifunctional polypeptide molecule comprises at least two cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises at least three cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises at least four cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises at least five cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises at least six cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises at least seven cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises at least eight cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises two cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises three cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises four cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises five cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises six cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises seven cytokine polypeptides. In some embodiments, a multifunctional polypeptide molecule comprises eight cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises two cytokine polypeptides, each linked to a first polypeptide and a second polypeptide; a first polypeptide and a third polypeptide; a first polypeptide and a fourth polypeptide; a second and a third polypeptide; a second polypeptide and a fourth polypeptide; or a third and a fourth polypeptide, respectively. In some embodiments, the multifunctional polypeptide molecule comprises three cytokine polypeptides, each linked to a first polypeptide, a second polypeptide, and a third polypeptide; a first polypeptide, a second polypeptide, and a fourth polypeptide; a first polypeptide, a third polypeptide, and a fourth polypeptide; or a second polypeptide, a third polypeptide, and a fourth polypeptide, respectively.In some embodiments, the multifunctional polypeptide molecule comprises four cytokine polypeptides, each linked to a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, respectively, hi some embodiments, the cytokine polypeptides are not linked to the polypeptide comprising the first TCRβV-binding portion.

[0229]

[0328] In some embodiments, the at least one cytokine polypeptide is selected from the group consisting of interleukin-2 (IL-2) or a fragment or functional fragment or functional variant thereof, or a combination thereof.

[0230]

[0329] In some embodiments, at least one cytokine polypeptide comprises interleukin-2 (IL-2) or a fragment thereof. In some embodiments, at least one cytokine polypeptide is interleukin-2 (IL-2) or a fragment thereof. In some embodiments, at least one cytokine polypeptide comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2191. In some embodiments, at least one cytokine polypeptide comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2191. In some embodiments, the sequence of at least one cytokine polypeptide is the sequence of SEQ ID NO: 2191.

[0231]

[0330] In some embodiments, the variant of at least one cytokine polypeptide comprises an IL-2 variant comprising a mutation. In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation. In some embodiments, the mutation comprises a substitution mutation. In some embodiments, the variant comprises an IL-2 variant comprising a C125A mutation. In some embodiments, the variant of at least one cytokine polypeptide is an IL-2 variant comprising a mutation. In some embodiments, the mutation is an insertion mutation, a deletion mutation, or a substitution mutation. In some embodiments, the mutation is a substitution mutation. In some embodiments, the variant is an IL-2 variant comprising a C125A mutation. In some embodiments, the variant comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2270. In some embodiments, the variant comprises the sequence of SEQ ID NO: 2270. In some embodiments, the sequence of the variant is a sequence that has at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 2270. In some embodiments, the sequence of the variant is the sequence of SEQ ID NO: 2270.

[0232]

[0331] In some embodiments, the first dimerization module comprises a first immunoglobulin constant region (Fc region) and the second dimerization module comprises a second Fc region. In some embodiments, the first dimerization module is a first immunoglobulin constant region (Fc region) and the second dimerization module is a second Fc region.

[0233]

[0332] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from an IgG1 Fc region or fragment thereof, an IgG2 Fc region or fragment thereof, an IgG3 Fc region or fragment thereof, an IgGA1 Fc region or fragment thereof, an IgGA2 Fc region or fragment thereof, an IgG4 Fc region or fragment thereof, an IgJ Fc region or fragment thereof, an IgM Fc region or fragment thereof, an IgD Fc region or fragment thereof, and an IgE Fc region or fragment thereof.

[0234]

[0333] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from a human IgG1 Fc region or fragment thereof, a human IgG2 Fc region or fragment thereof, and a human IgG4 Fc region or fragment thereof.

[0235]

[0334] In some embodiments, the first Fc region, the second Fc region, or a combination thereof, comprises an Fc interface having one or more of a cavity-protrusion pair, an electrostatic interaction, or strand exchange, and dimerization of the first Fc region and the second Fc region is enhanced as indicated by a higher ratio of heteromultimers:homomultimers compared to dimerization of an Fc region having an unengineered interface. In some embodiments, the dimerization of the first Fc region and the second Fc region is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, or greater than the dimerization of an Fc region having an unengineered interface. , 70x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 200x, 250x, 300x, 250x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, or 10000x. In some embodiments, the dimerization of the first Fc region and the second Fc region is at most 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, and / or 10,000-fold enhanced by 70x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 200x, 250x, 300x, 250x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, or 10,000x.In some embodiments, the dimerization of the first Fc region and the second Fc region is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75 ... Enhanced by 0x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 200x, 250x, 300x, 250x, 400x, 450x, 500x, 550x, 600x, 650x, 700x, 750x, 800x, 850x, 900x, 950x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, or 10000x.

[0236]

[0335] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an amino acid substitution set forth in Table 14.

[0336] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Asn297Ala (N297A) mutation or a Leu234Ala / Leu235Ala (LALA) mutation.

[0237]

[0337] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649. In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649.

[0238]

[0338] In some embodiments, the sequence of the first Fc region, the second Fc region, or a combination thereof is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649. In some embodiments, the sequence of the first Fc region, the second Fc region, or a combination thereof is the sequence of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 3645, SEQ ID NO: 3646, SEQ ID NO: 3647, SEQ ID NO: 3648, or SEQ ID NO: 3649.

[0239]

[0339] In some embodiments, the first TCRβ V binding moiety, the second TCRβ V binding moiety, or a combination thereof, is (i) TCRβ V6-4 * 01. TCRβ V6-4 * 02. TCRβ V6-9 * 01. TCRβ V6-8 * 01. TCRβ V6-5 * 01. TCRβ V6-6 * 02. TCRβ V6-6 * 01. TCRβ V6-2 * 01. TCRβ V6-3 * 01, and TCRβ V6-1 * 01, and (ii) TCRβ V10-1 * 01. TCRβ V10-1 * 02. TCRβ V10-3 * 01, and TCRβ V10-2 * 01.

[0240]

[0340] In some embodiments, the first TCRβV binding moiety and the second TCRβV binding moiety are the same. In some embodiments, the first TCRβV binding moiety and the second TCRβV binding moiety are different.

[0241]

[0341] In some embodiments, the first TCRβV binding moiety and the second TCRβV binding moiety bind to one or more TCRβ V6 subfamily members and one or more TCRβ V10 subfamily members, respectively.

[0242]

[0342] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises (i) HC CDR1, HC CDR2, and HC CDR3 whose amino acid sequences have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with any one of the CDR1, CDR2, and CDR3 sequences set forth in Table 1; (ii) LC CDR1, LC CDR2, and LC CDR3 whose amino acid sequences have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with any one of the CDR1, CDR2, and CDR3 sequences set forth in Table 1; or (iii) a combination thereof. In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises (i) an HC CDR1, an HC CDR2, and an HC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences set forth in Table 1; (ii) an LC CDR1, an LC CDR2, and an LC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences set forth in Table 1; or (iii) a combination thereof.

[0243]

[0343] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises (i) HC CDR1, HC CDR2, and HC CDR3 whose amino acid sequences have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1, respectively; (ii) LC CDR1, LC CDR2, and LC CDR3 whose amino acid sequences have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1, respectively; or (iii) a combination thereof. In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises (i) an HC CDR1, an HC CDR2, and an HC CDR3, each having any one of the CDR1, CDR2, and CDR3 sequences set forth in Table 1; (ii) an LC CDR1, an LC CDR2, and an LC CDR3, each having any one of the CDR1, CDR2, and CDR3 sequences set forth in Table 1; or (iii) a combination thereof.

[0244]

[0344] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof, comprises (i) framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and / or a non-mouse germline FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4. 3), and a framework region (FR) comprising framework region 4 (FR4); (ii) a VL comprising FRs comprising a non-mouse germline FR1, a non-mouse germline FR2, a non-mouse germline FR3, and a FR1, FR2, FR3, and FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the non-mouse germline FR4; or (iii) a combination thereof. In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises: (i) a VH comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4; (ii) a VL comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4; or (iii) a combination thereof.

[0245]

[0345] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises: (i) a VH comprising FR1, FR2, FR3, and FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; (ii) a VL comprising FRs that comprise FR1, FR2, FR3, and FR4 that have at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity with non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; or (iii) a combination thereof. In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises: (i) a VH comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; (ii) a VL comprising FRs comprising FR1, FR2, FR3, and FR4 having the sequences of non-mouse germline FR1, non-mouse germline FR2, non-mouse germline FR3, and non-mouse germline FR4, respectively; or (iii) a combination thereof.

[0246]

[0346] In some embodiments, the VH comprises an FR3 comprising: (i) a threonine at position 73 according to Kabat numbering; (ii) a glycine at position 94 according to Kabat numbering; or (iii) a combination thereof. In some embodiments, the VL comprises an FR1 comprising a phenylalanine at position 10 according to Kabat numbering. In some embodiments, the VL comprises an FR2 comprising: (i) a histidine at position 36 according to Kabat numbering; (ii) an alanine at position 46 according to Kabat numbering; or (iii) a combination thereof. In some embodiments, the VL comprises an FR3 comprising a phenylalanine at position 87 according to Kabat numbering.

[0247]

[0347] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain variable region having a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 3, or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having any one of the sequences listed in Table 3, or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region whose sequence has at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences listed in Table 3, or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region having any one of the heavy chain constant regions set forth in Table 3, or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises an IgM heavy chain constant region, or a fragment thereof. In some embodiments, the IgM heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 73. In some embodiments, the IgM heavy chain constant region comprises the sequence of SEQ ID NO: 73. In some embodiments, the sequence of the IgM heavy chain constant region is the sequence of SEQ ID NO: 73.

[0248]

[0348] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises an IgJ heavy chain constant region or a fragment thereof. In some embodiments, the IgJ heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments, the IgJ heavy chain constant region comprises the sequence of SEQ ID NO: 76. In some embodiments, the sequence of the IgJ heavy chain constant region is the sequence of SEQ ID NO: 76.

[0249]

[0349] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of IgGA1 or a fragment thereof. In some embodiments, the heavy chain constant region of IgGA1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 74. In some embodiments, the heavy chain constant region of IgGA1 comprises the sequence of SEQ ID NO: 74. In some embodiments, the sequence of the heavy chain constant region of IgGA1 is the sequence of SEQ ID NO: 74.

[0250]

[0350] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a heavy chain constant region of IgGA2 or a fragment thereof. In some embodiments, the heavy chain constant region of IgGA2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, the heavy chain constant region of IgGA2 comprises the sequence of SEQ ID NO: 75. In some embodiments, the sequence of the heavy chain constant region of IgGA2 is the sequence of SEQ ID NO: 75.

[0251]

[0351] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises an IgG1 heavy chain constant region or a fragment thereof. In some embodiments, the IgG1 heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 41. In some embodiments, the IgG1 heavy chain constant region comprises the sequence of SEQ ID NO: 41. In some embodiments, the sequence of the IgG1 heavy chain constant region is the sequence of SEQ ID NO: 41. In some embodiments, the IgG1 heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 3645. In some embodiments, the IgG1 heavy chain constant region comprises the sequence of SEQ ID NO: 3645. In some embodiments, the sequence of the heavy chain constant region of IgG1 is the sequence of SEQ ID NO:3645.

[0252]

[0352] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the sequences set forth in Table 3, or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having any one of the sequences set forth in Table 3, or a combination thereof. In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region having any one of the light chain constant region sequences set forth in Table 3, or a combination thereof.

[0253]

[0353] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises a light chain constant region of a kappa chain or a fragment thereof. In some embodiments, the light chain constant region of a kappa chain comprises a light chain constant region sequence set forth in Table 3.

[0254]

[0354] In some embodiments, the light chain constant region of the kappa chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the light chain constant region of the kappa chain comprises the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the sequence of the light chain constant region of the kappa chain is a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644. In some embodiments, the sequence of the light chain constant region of the kappa chain is the sequence of SEQ ID NO: 39 or SEQ ID NO: 3644.

[0255]

[0355] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof, comprises a light chain comprising an FR1 comprising: (i) an aspartic acid at position 1 according to Kabat numbering; (ii) an asparagine at position 2 according to Kabat numbering; (iii) a leucine at position 4 according to Kabat numbering; or (iv) a combination thereof.

[0256]

[0356] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof, comprises a light chain comprising an FR3 comprising: (i) a glycine at position 66 according to Kabat numbering; (ii) an asparagine at position 69 according to Kabat numbering; (iii) a tyrosine at position 71 according to Kabat numbering; or (iv) a combination thereof.

[0257]

[0357] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof binds to an outer-facing region of the TCRβV protein. In some embodiments, the outer-facing region of the TCRβV protein comprises a structurally conserved region of TCRβV that has a similar structure among one or more TCRβV subfamilies. Cytokine molecules

[0358] In some embodiments, the multifunctional molecule comprises a cytokine molecule. As used herein, "cytokine molecule" or "cytokine polypeptide" are used interchangeably herein and refer to a full-length, fragment, or variant of a cytokine; a cytokine that further comprises a receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonist antibody) against a cytokine receptor, that induces at least one activity of a naturally occurring cytokine. In some embodiments, the cytokine molecule is interleukin-2 (IL-2), or a fragment or variant thereof, or a combination thereof. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule can further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor.

[0258]

[0359] Cytokines are generally polypeptides that affect cellular activity, for example, through signal transduction pathways. Thus, multispecific or multifunctional polypeptide cytokines are useful and can be involved in receptor-mediated signal transduction, transmitting signals from the outside of the cell membrane to modulate intracellular responses. Cytokines are proteinaceous signaling compounds that mediate immune responses. They control many different cellular functions, including proliferation, differentiation, and cell survival / apoptosis; cytokines are also involved in several pathophysiological processes, including viral infections and autoimmune diseases. Cytokines are synthesized under various stimuli by various cells of both the innate immune system (monocytes, macrophages, dendritic cells) and the adaptive immune system (T cells and B cells). Cytokines can be classified into two groups: pro-inflammatory and anti-inflammatory. Pro-inflammatory cytokines include IFNγ, IL-1, IL-6, and TNF-alpha and are primarily derived from innate and Th1 cells. Anti-inflammatory cytokines include IL-10, IL-4, IL-13, and IL-5 and are synthesized by Th2 immune cells.

[0259]

[0360] Provided herein, among other things, are multispecific (e.g., bi-, tri-, tetraspecific) or multifunctional molecules that comprise, e.g., are engineered to contain, one or more cytokine molecules, e.g., immunomodulatory (e.g., pro-inflammatory) cytokines and variants, e.g., functional variants thereof. Thus, in some embodiments, the cytokine molecule is an interleukin or a variant, e.g., a functional variant, thereof. In some embodiments, the interleukin is a pro-inflammatory interleukin. In some embodiments, the interleukin is interleukin-2 (IL-2). In some embodiments, the cytokine molecule is a pro-inflammatory cytokine.

[0260]

[0361] In certain embodiments, the cytokine is a single-chain cytokine. In certain embodiments, the cytokine is a multi-chain cytokine (e.g., a cytokine comprising two or more (e.g., two) polypeptide chains).

[0261]

[0362] Examples of useful cytokines include, but are not limited to, IL-2. In some embodiments, the cytokine of the multispecific or multifunctional polypeptide is IL-2. In certain embodiments, the cytokine is mutated to remove N- and / or O-glycosylation sites. Removal of glycosylation increases the homogeneity of the product that can be obtained in recombinant production.

[0262]

[0363] In some embodiments, the cytokine of the multispecific or multifunctional polypeptide is IL-2. In certain embodiments, the IL-2 cytokine can induce one or more cellular responses selected from the group consisting of proliferation of activated T lymphocyte cells, differentiation of activated T lymphocyte cells, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation of activated B cells, proliferation of natural killer (NK) cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) anti-tumor cytotoxicity. In another specific embodiment, the IL-2 cytokine is a mutant IL-2 cytokine having reduced binding affinity for the alpha-subunit of the IL-2 receptor. The alpha-subunit (also known as CD25) together with the beta- and gamma-subunits (also known as CD122 and CD132, respectively) form a heterodimeric high-affinity IL-2 receptor, while a dimeric receptor consisting only of the beta- and gamma-subunits is referred to as an intermediate-affinity IL-2 receptor. As described in PCT Patent Application No. PCT / EP2012 / 051991, the entirety of which is incorporated herein by reference, mutant IL-2 polypeptides with reduced binding to the alpha-subunit of the IL-2 receptor have reduced ability to induce IL-2 signaling in regulatory T cells, induce less activation-induced cell death (AICD) in T cells, and have a reduced toxicity profile in vivo compared to wild-type IL-2 polypeptides. The use of such cytokines with reduced toxicity is particularly advantageous in multispecific or multifunctional polypeptides in accordance with the present invention that have a long serum half-life due to the presence of an Fc domain. In some embodiments, a mutant IL-2 cytokine in a multispecific or multifunctional polypeptide in accordance with the present invention comprises at least one amino acid mutation that reduces or eliminates the affinity of the mutant IL-2 cytokine for the alpha-subunit of the IL-2 receptor (CD25) compared to the unmutated IL-2 cytokine, while preserving the affinity of the mutant IL-2 cytokine for the intermediate-affinity IL-2 receptor (consisting of the β and γ subunits of the IL-2 receptor).In some embodiments, the one or more amino acid mutations are amino acid substitutions. In specific embodiments, the mutant IL-2 cytokine contains one, two, or three amino acid substitutions at one, two, or three positions selected from those corresponding to residues 42, 45, and 72 of human IL-2. In more specific embodiments, the mutant IL-2 cytokine contains three amino acid substitutions at positions corresponding to residues 42, 45, and 72 of human IL-2. In even more specific embodiments, the mutant IL-2 cytokine is human IL-2 containing the amino acid substitutions F42A, Y45A, and L72G. In some embodiments, the mutant IL-2 cytokine further contains an amino acid mutation that eliminates the O-glycosylation site of IL-2 at a position corresponding to position 3 of human IL-2. In particular, the additional amino acid mutation is an amino acid substitution that replaces a threonine residue with an alanine residue. A particular mutant IL-2 cytokine useful in the present invention contains four amino acid substitutions at positions corresponding to residues 3, 42, 45, and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A, and L72G. As demonstrated in PCT Patent Application No. PCT / EP2012 / 051991 and the accompanying Examples, the quadruple mutant IL-2 polypeptide (IL-2 qm) exhibits no detectable binding to CD25, exhibits reduced ability to induce apoptosis in T cells, exhibits reduced ability to induce IL-2 signaling in T.sub.reg cells, and exhibits a reduced toxicity profile in vivo. However, it retains the ability to activate IL-2 signaling in effector cells, induce effector cell proliferation, and produce IFN-γ as a secondary cytokine by NK cells.

[0263]

[0364] The IL-2 or mutant IL-2 cytokine according to any of the above embodiments may contain additional mutations that provide additional advantages, such as increased expression or stability. For example, to avoid the formation of disulfide-bridged IL-2 dimers, the cysteine ​​at position 125 may be replaced with a neutral amino acid, such as alanine. Thus, in certain embodiments, the IL-2 or mutant IL-2 cytokine of a multispecific or multifunctional polypeptide according to the present invention contains an additional amino acid mutation at the position corresponding to residue 125 of human IL-2. In some embodiments, the additional amino acid mutation is the amino acid substitution C125A.

[0264]

[0365] In a specific embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide comprises the polypeptide sequence of SEQ ID NO:2270. [APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT]

[0366] In another specific embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide comprises the polypeptide sequence of SEQ ID NO:2280. [APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT]

[0367] Mutant cytokine molecules useful as effector moieties in multispecific or multifunctional polypeptides can be prepared by deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. Correct nucleotide changes can be confirmed, for example, by sequencing. Substitutions or insertions can involve natural and unnatural amino acid residues. Amino acid modifications include well-known chemical modification methods such as adding or removing glycosylation sites or attaching sugar chains, and the like.

[0265]

[0368] In some embodiments, the multispecific or multifunctional polypeptides of the invention have a dissociation constant (K D ) at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times higher K D In another embodiment, the multispecific or multifunctional polypeptide binds to a cytokine receptor at a K of a corresponding multispecific or multifunctional polypeptide comprising two or more effector moieties. D at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than D In another embodiment, the multispecific or multifunctional polypeptide binds to a cytokine receptor at a K of the corresponding multispecific or multifunctional polypeptide comprising two or more cytokines. D Approximately 10 times higher dissociation constant K D binds to cytokine receptors.

[0266]

[0369] In some embodiments, the multispecific molecules described herein comprise a cytokine molecule. In embodiments, the cytokine molecule comprises a full-length, fragment, or variant of a cytokine; a cytokine receptor domain, e.g., a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule against a cytokine receptor (e.g., an agonist antibody).

[0267]

[0370] In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2 (e.g., the amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2191), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence that is 95% to 99.9% identical thereto, or that has at least one amino acid change relative to the amino acid sequence of SEQ ID NO: 2191, but has no more than 5, 10, or 15 changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions)).

[0268] Immune Cell Engager

[0371] In some embodiments, the multifunctional molecule further comprises an immune cell engager. "Immune cell engager" refers to one or more binding specificities that bind to and / or activate immune cells, e.g., cells involved in an immune response. In embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, and / or macrophage cells. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full-length receptor, a receptor fragment, or a fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full-length ligand, a ligand fragment, or a fusion thereof (e.g., a ligand-Fc fusion)) that binds to an immune cell antigen (e.g., a T cell, NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen). In embodiments, the immune cell engager specifically binds to a target immune cell, e.g., preferentially binds to a target immune cell. For example, if the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, an NK cell antigen, a B cell antigen, a dendritic cell antigen, and / or a macrophage cell antigen) with a dissociation constant of less than about 10 nM.

[0269]

[0372] The immune cell engager, e.g., the first and / or second immune cell engager of the multispecific or multifunctional molecule described herein, can mediate binding to and / or activation of an immune cell, e.g., an immune effector cell. In some embodiments, the immune cell is selected from a T cell, an NK cell, a B cell, a dendritic cell, or a macrophage cell engager, or a combination thereof. In some embodiments, the immune cell engager is selected from one, two, three, or all of a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager, or a combination thereof. The immune cell engager can be an agonist of the immune system. In some embodiments, the immune cell engager can be an antibody molecule, a ligand molecule (e.g., a ligand further comprising an immunoglobulin constant region, e.g., an Fc region), a small molecule, or a nucleotide molecule.

[0270] antibody molecule

[0373] In some embodiments, the antibody molecule binds to a cancer antigen, such as a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, for example, a mammalian, e.g., human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, such as a mammalian, e.g., human, immune cell antigen. For example, the antibody molecule specifically binds to an epitope, such as a linear or conformational epitope, on the cancer antigen or immune cell antigen.

[0271]

[0374] In some embodiments, the antibody molecule is a monospecific antibody molecule, which binds to a single epitope, e.g., a monospecific antibody molecule has multiple immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0272]

[0375] In some embodiments, the antibody molecule is a multispecific or multifunctional antibody molecule, e.g., comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has specific binding to a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity to a second epitope. In some embodiments, the first and second epitopes are present on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are present on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In some embodiments, the multifunctional antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In some embodiments, the multifunctional antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0273]

[0376] In some embodiments, the multifunctional antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are present on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are present on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope, and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, that has binding specificity for a first epitope, and a half antibody, or fragment thereof, that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv or Fab, or fragment thereof, that has binding specificity for a first epitope, and the scFv or Fab, or fragment thereof, has binding specificity for a second epitope.

[0274]

[0377] In some embodiments, antibody molecules include diabodies and single-chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule may comprise a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as half antibodies). In another example, an antibody molecule comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of whole antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be derived from any class of antibody, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted, or in vitro-generated. Antibodies can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies can also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein.

[0275]

[0378] Examples of antigen-binding fragments of antibody molecules include: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camel or camelized variable domain; (vii) a single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); and (viii) a single-domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0276]

[0379] Antibody molecules include intact molecules as well as functional fragments thereof. The constant region of an antibody molecule can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0277]

[0380] The antibody molecule may also be a single-domain antibody. Single-domain antibodies may include antibodies whose complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be any antibody in the art or any future single-domain antibody. Single-domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another embodiment of the present invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking light chains. Such single-domain antibodies are disclosed, for example, in WO 9404678. For clarity, this variable domain derived from a heavy-chain antibody naturally lacking light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other non-Camelidae species may produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present invention.

[0278]

[0381] The VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity-determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FR or FW).

[0279]

[0382] The extent of framework regions and CDRs has been precisely defined by several methods (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software).In general, see, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains.In:Antibody Engineering Lab Manual (Ed.:Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0280]

[0383] The terms "complementarity determining region" and "CDR" as used herein refer to the amino acid sequences in an antibody variable region that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0281]

[0384] The precise amino acid sequence boundaries of a given CDR can be determined using any of several known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme are also sometimes referred to as "hypervariable loops."

[0282]

[0385] For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0283]

[0386] Each VH and VL typically comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0284]

[0387] The antibody molecule may be a polyclonal or a monoclonal antibody.

[0388] The terms "monoclonal antibody" or "monoclonal antibody composition," as used herein, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies may be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0285]

[0389] Antibodies can be produced recombinantly, for example, by phage display, or combinatorial methods, or yeast display.

[0390] Phage display and combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. WO 92 / 18619; Dower et al. WO 91 / 17271; Winter et al. WO 92 / 20791; Markland et al. WO 92 / 15679; Breitling et al. WO 93 / 01288; McCafferty et al. WO 92 / 01047; Garrard et al. WO 92 / 09690; Ladner et al. WO 90 / 02809; Fuchs et al., the entire contents of which are incorporated herein by reference). al.(1991)Bio / Technology 9:1370-1372;Hay et al.(1992)Hum Antibod Hybridomas 3:81-85;Huse et al.(1989)Science 246:1275-1281;Griffths et al.(1993)EMBO J 12:725-734;Hawkins et al. al.(1992)J Mol Biol 226:889-896;Clackson et al.(1991)Nature 352:624-628;Gram et al.(1992)PNAS 89:3576-3580;Garrad et al.(1991)Bio / Technology 9:1373-1377;Hoogenboom et al. al.(1991)Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982).

[0286]

[0391] Yeast display for generating or identifying antibodies is known in the art, for example, as described in Chao et al. (2006) Nature Protocols 1(2):755-68, the entire contents of which are incorporated herein by reference.

[0287]

[0392] In some embodiments, the antibody is a fully human antibody (e.g., an antibody made in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0288]

[0393] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes from human proteins (see, e.g., Wood et al. WO 91 / 00906; Kucherlapati et al. PCT Publication WO 91 / 10741; Lonberg et al. WO 92 / 03918; Kay et al. WO 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0289]

[0394] The antibody molecule may be an antibody molecule whose variable region, or a part thereof, such as CDR, is produced in a non-human organism, such as a rat or a mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. Antibody molecules produced in a non-human organism, such as a rat or a mouse, and then modified, for example, in the variable framework region or constant region, to reduce antigenicity in humans, are within the scope of the present invention.

[0290]

[0395] A "effectively human" protein is one that does not substantially induce a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be a problem in several situations, for example, when antibody molecules are administered repeatedly, for example, in the treatment of chronic or recurring disease states. HAMA responses can potentially render repeated administration of antibodies ineffective due to increased antibody clearance from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0291]

[0396] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al. PCT / US86 / 02269; Akira, et al. European Patent Application No. 184,187; Taniguchi, M. European Patent Application No. 171,496; Morrison et al. European Patent Application No. 173,494; Neuberger et al. International Patent Application WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application No. 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun (See, e.g., Nishimura et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0292]

[0397] Humanized or CDR-grafted antibodies have at least one or two, but generally all three, recipient CDRs (of the immunoglobulin heavy and / or light chain) replaced with donor CDRs. The antibody may have at least a portion of the non-human CDRs replaced, or only a portion of the CDRs replaced with non-human CDRs. It is only necessary to replace as many CDRs as necessary for antigen binding. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "acceptor." In some embodiments, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is about 85% or more, preferably 90%, 95%, 99% or more identical thereto.

[0293]

[0398] As used herein, the term "consensus sequence" refers to the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a protein family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. "Consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

[0294]

[0399] Antibody molecules may be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, the entire contents of which are hereby incorporated by reference).

[0295]

[0400] Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR-substitution, in which one, two, or all CDRs of an immunoglobulin chain can be replaced. See, for example, U.S. Patent No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter, U.S. Patent No. 5,225,539, the entire contents of which are hereby incorporated by reference. Winter describes a CDR-grafting method that can be used to prepare the humanized antibodies of the present invention (UK Patent Application No. 2188638A, filed March 26, 1987; Winter, U.S. Patent No. 5,225,539, the contents of which are hereby incorporated by reference).

[0296]

[0401] Similarly, humanized antibody molecules in which specific amino acids have been substituted, deleted, or added are also within the scope of the present invention. Criteria for selecting amino acids from the donor are described in U.S. Patent No. 5,585,089, e.g., columns 12-16 of U.S. Patent No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al., European Patent No. 519596 A1, filed December 23, 1992.

[0297]

[0402] The antibody molecule can be a single-chain antibody. Single-chain antibodies (scFV) can be engineered (see, for example, Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Single-chain antibodies can be dimerized or multimerized to generate multivalent antibodies with specificity for different epitopes of the same target protein.

[0298]

[0403] In still other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, for example, the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, and / or complement function). In some embodiments, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells or fix complement. In another embodiment, the antibody has reduced or no Fc receptor binding ability. For example, it may be an isotype or subtype, fragment, or other variant that does not support binding to an Fc receptor, eg, has a mutated or deleted Fc receptor binding region.

[0299]

[0404] Methods for modifying antibody constant regions are known in the art.Antibodies with altered functions, such as altered affinity for effector ligands, such as FcR on cells, or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, for example, European Patent No. 388,151A1, U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260, the entire contents of which are hereby incorporated by reference).Similar types of modifications can be described that, when applied to immunoglobulins of mice or other species, reduce or eliminate these functions.

[0300]

[0405] Antibody molecules can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is a molecule that has been modified. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radionuclide, a toxin, an enzyme, or an affinity ligand such as biotin. Thus, the antibody molecules of the present invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a cytotoxic agent, a drug, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0301]

[0406] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0302] CDR graft scaffold

[0407] In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, for example, a fibronectin type III domain. The overall folding of the fibronectin type III (Fn3) domain is closely related to that of the smallest functional antibody fragment, the variable domain of an antibody heavy chain. There are three loops at the end of Fn3; the positions of the BC, DE, and FG loops roughly correspond to the positions of CDR1, 2, and 3 of the VH domain of an antibody. Fn3 does not have disulfide bonds; therefore, unlike antibodies and their fragments, Fn3 is stable under reducing conditions (see, for example, WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). The Fn3 domain can be modified (e.g., using the CDRs or hypervariable loops described herein) or altered, for example, to select domains that bind to the antigens / markers / cells described herein.

[0303]

[0408] In some embodiments, the scaffold domain, e.g., the folded domain, is based on a "minibody" scaffold created by deleting three beta strands from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al., 1994, J. Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). A "minibody" can be used to display two hypervariable loops. In some embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al., WO 99 / 45110) or a domain derived from tendamistatin, a 74-residue six-stranded beta-sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J. Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified (e.g., using CDRs or hypervariable loops) or altered to select domains that bind to, for example, the markers / antigens / cells described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00 / 60070).

[0304]

[0409] Other exemplary scaffolding domains include, but are not limited to, T cell receptors; MHC proteins; extracellular domains (e.g., fibronectin type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and others); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA-binding proteins; RNA-binding proteins; enzymes, such as proteases (particularly inactivating proteases), RNases; chaperones, such as thioredoxin and heat shock proteins; and intracellular signaling domains (e.g., SH2 and SH3 domains). See, e.g., U.S. Patent Application Publication No. 20040009530 and U.S. Patent No. 7,501,121, which are incorporated herein by reference.

[0305]

[0410] In some embodiments, scaffold domains are evaluated and selected by one or more of the following criteria: (1) amino acid sequence, (2) sequence of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salt concentration, organic solvent, and oxidant concentration. In some embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40, or 30 amino acids. The domain may contain one or more disulfide bonds or may chelate a metal, e.g., zinc.

[0306] Antibody-Based Fusions

[0411] A variety of formats can be produced containing additional binding entities attached to the N- or C-terminus of the antibody. Fusion of these with single-chain or disulfide-stabilized Fv or Fab results in the generation of tetravalent molecules with bivalent binding specificities for each antigen. Combining scFv and scFab with IgG allows the production of molecules capable of recognizing three or more different antigens.

[0307] Antibody-Fab fusion

[0412] An antibody-Fab fusion is a bispecific antibody that contains a conventional antibody against a first target and a Fab against a second target fused to the C-terminus of the antibody heavy chain. Generally, the antibody and Fab share a common light chain. Antibody fusions can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. Antibody-scFv fusions appear to be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described in Coloma, J. et al. (1997) Nature Biotech 15:159.

[0308] Antibody-scFv fusion

[0413] Antibody-scFv fusions are bispecific antibodies containing a conventional antibody and a uniquely specific scFv fused to the C-terminus of the antibody heavy chain. The scFv can be fused directly to the heavy chain of the scFv or to the C-terminus via a linker peptide. Antibody fusions can be produced by (1) manipulating the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. Antibody-scFv fusions appear to be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described in Coloma, J. et al. (1997) Nature Biotech 15:159. Variable Domain Immunoglobulin DVD

[0414] A related format is the dual variable domain immunoglobulin (DVD), which consists of a VH and VL domain of a second specificity positioned N-terminal to the V domain by a shorter linker sequence.

[0309]

[0415] Other exemplary multifunctional antibody formats include, for example, U.S. Patent Application Publication No. 20160114057A1, U.S. Patent Application Publication No. 20130243775A1, U.S. Patent Application Publication No. 20140051833, U.S. Patent Application Publication No. 20130022601, U.S. Patent Application Publication No. 20150017187A1, U.S. Patent Application Publication No. 20120201746A1, U.S. Patent Application Publication No. 20150133638A1, U.S. Patent Application Publication No. 20130266568A1, U.S. Patent Application Publication No. 20160145340A1, International Publication No. 2015127158A1, U.S. Patent Application Publication No. 20150203591A1, U.S. Patent Application Publication No. 20140322221A1 No. 20130303396A1, U.S. Patent Application Publication No. 20110293613, U.S. Patent Application Publication No. 20130017200A1, U.S. Patent Application Publication No. 20160102135A1, WO 2015197598A2, WO 2015197582A1, U.S. Patent No. 9,359,437, U.S. Patent Application Publication No. 20150018529, WO 2016115274A1, WO 2016087416A1, U.S. Patent Application Publication No. 20080069820A1, U.S. Patent No. 9,145,588B, U.S. Patent No. 7,919,257, and U.S. Patent Application Publication No. 20150232560A1. Exemplary multifunctional molecules utilizing the whole antibody-Fab / scFab format include those described in the following: U.S. Patent No. 9,382,323 B2, U.S. Patent Application Publication No. 20140072581 A1, U.S. Patent Application Publication No. 20140308285 A1, U.S. Patent Application Publication No. 20130165638 A1, U.S. Patent Application Publication No. 20130267686 A1, U.S. Patent Application Publication No. 20140377269 A1, U.S. Patent No. 7,741,446 B2, and WO 1995009917 A1.Exemplary multifunctional molecules utilizing the domain-swapping format include those described in the following: U.S. Patent Application Publication No. 20150315296A1, WO 2016087650A1, U.S. Patent Application Publication No. 20160075785A1, WO 2016016299A1, U.S. Patent Application Publication No. 20160130347A1, U.S. Patent Application Publication No. 20150166670, U.S. Patent No. 8703132B2, U.S. Patent Application Publication No. 20100316645, U.S. Patent No. 8227577B2, U.S. Patent Application Publication No. 20130078249.

[0310] Fc-containing multifunctional molecules

[0416] In some embodiments, the multifunctional molecules described herein comprise an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4; more particularly, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0311]

[0417] In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0312]

[0418] In other embodiments, the interface of the first and second immunoglobulin chain constant regions (e.g., first and second Fc regions) is altered, e.g., mutated, to increase or decrease dimerization compared to an unengineered interface, e.g., a naturally occurring interface. For example, dimerization of immunoglobulin chain constant regions (e.g., Fc regions) can be enhanced by providing an interface of the first and second Fc regions with one or more of a knob-in-hole pair, electrostatic interactions, or chain exchange, such that the ratio of heteromultimers to homomultimers is greater compared to the unengineered interface.

[0313]

[0419] In some embodiments, the multifunctional molecule comprises a pair of amino acid substitutions at a position selected from one or more of positions 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region, e.g., of human IgG1. For example, the immunoglobulin chain constant region (e.g., Fc region) can comprise a pair of amino acid substitutions selected from T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), and T366W (e.g., corresponding to a protrusion or knob).

[0314]

[0420] In other embodiments, the multifunctional molecule comprises a half-life extender, for example, human serum albumin or an antibody molecule against human serum albumin.

[0421] In some embodiments, the Fc contains the exemplary Fc modifications set forth in Table 14.

[0315] Heterodimerized antibody molecules and methods of production

[0422] Various methods for producing multifunctional antibodies have been disclosed to address the problem of inaccurate heavy chain pairing. Exemplary methods are described below. Exemplary multifunctional antibody formats and methods for producing said multifunctional antibodies are also disclosed, for example, in Speiss et al. Molecular Immunology 67 (2015) 95-106; and Klein et al. mAbs 4:6, 653-663; November / December 2012, the entire contents of each of which are incorporated herein by reference.

[0316]

[0423] Heterodimerized bispecific antibodies are based on the natural IgG structure, with the two binding arms recognizing different antigens. IgG-derived formats that allow for defined monovalent (and parallel) antigen binding are generated by forced heavy chain heterodimerization combined with techniques that minimize light chain mispairing (e.g., common light chains). Forced heavy chain heterodimerization can be achieved, for example, using knobs-in-holes or strand-exchange engineered domains (SEEDs).

[0317] Nobu in Hall

[0424] Knobs-in-holes, as described in U.S. Patent Nos. 5,731,116, 7,476,724, and Ridgway, J. et al. (1996) Prot. Engineering 9(7):617-621, broadly involve (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization, and (2) combining the mutated antibodies under conditions that promote heterodimerization. The "knob" or "protrusion" is typically created by replacing a small amino acid in the parent antibody with a larger one (e.g., T366Y or T366W); the "hole" or "cavity" is created by replacing a larger residue in the parent antibody with a smaller one (e.g., Y407T, T366S, L368A, and / or Y407V).

[0318]

[0425] For bispecific antibodies containing an Fc domain, specific mutations can be introduced into the constant region of the heavy chain to promote correct heterodimerization of the Fc portion. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the "knob-into-hole" (KiH) approach, which involves introducing bulky residues into one CH3 domain of one antibody heavy chain. This bulky residue fits into a complementary "hole" in the CH3 domain of the other paired heavy chain, promoting correct pairing of the heavy chains (see, for example, U.S. Patent No. 7,642,228).

[0319]

[0426] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Other exemplary KiH mutations, along with additional optional stabilizing Fc cysteine ​​mutations, are provided in Table 4.

[0320]

[0427] Another Fc mutation was provided by Igawa and Tsunoda, who identified three negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are E356-K439, E357-K370, D399-K409, and vice versa. By introducing at least two of the following three mutations in chain A: E356K, E357K, and D399K, and at least two of the following mutations in chain B: K370E, K409D, and K439E, either alone or in combination with newly identified disulfide bridges, they can support highly efficient heterodimerization while simultaneously suppressing homodimerization (Martens T et al. A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006;12:6144-52; PMID:17062691). Xencor defined 41 variant pairs based on a combination of structural calculations and sequence information, then screened for maximum heterodimerization, identifying the combinations S364H, F405A for chain A (HA) and Y349T, T394F for chain B (TF) (Moore GL et al. A novel bispecfic antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011;3:546-57; PMID:22123055).

[0321]

[0428] Other exemplary Fc mutations to promote heterodimerization of multifunctional antibodies include those described in the following references, the contents of each of which are incorporated herein by reference: WO 2016071377A1, U.S. Patent Application Publication No. 20140079689A1, U.S. Patent Application Publication No. 20160194389A1, U.S. Patent Application Publication No. 20160257763, International Publication No. 2016071376A2, International Publication No. 2015107026A1, International Publication No. 2015107025A1, International Publication No. 2015107015A1, U.S. Patent Application Publication No. 20150353636A1, U.S. Patent Application Publication No. 20140199294A1, U.S. Patent No. 7750128B2, U.S. Patent Application Publication No. 20160229915A1, U.S. Patent Application Publication No. 20150344570A1, U.S. Patent No. 8003774A1, U.S. Patent Application Publication No. 20150337049A1, U.S. Patent Application Publication No. 20150175707A1, U.S. Patent Application Publication No. 20140242075A1, U.S. Patent Application Publication No. 20130195849A1, U.S. Patent Application Publication No. 20120149876A1, U.S. Patent Application Publication No. No. 20140200331A1, U.S. Patent No. 9309311B2, U.S. Patent No. 8586713, U.S. Patent Application Publication No. 20140037621A1, U.S. Patent Application Publication No. 20130178605A1, U.S. Patent Application Publication No. 20140363426A1, U.S. Patent Application Publication No. 20140051835A1 and U.S. Patent Application Publication No. 20110054151A1.

[0322]

[0429] Stabilizing cysteine ​​mutations have also been used in combination with KiH and other Fc heterodimerization-promoting variants.See, for example, U.S. Patent No. 7,183,076.Other exemplary cysteine ​​modifications include those disclosed in, for example, U.S. Patent Application Publication No. 20140348839A1, U.S. Patent No. 7,855,275B2, and U.S. Patent No. 9,000,130B2.

[0323] Strand exchange engineering domain (SEED)

[0430] A heterodimeric Fc platform is known that supports the design of bispecific and asymmetric fusion proteins by designing strand-exchange engineered domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEED body (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3], which can be genetically linked to one or more fusion partners (see, e.g., Davis JH et al. SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng Des Sel 2010;23:195-202; PMID:20299542 and US8871912, the contents of each of which are incorporated herein by reference).

[0324] Fc-containing entities (miniantibodies)

[0431] Fc-containing entities, also known as miniantibodies, can be generated by fusing scFv to the C-terminus of the constant heavy chain domain 3 (CH3-scFv) and / or hinge region (scFv-hinge-Fc) of an antibody with a different specificity. Trivalent entities can also be made in which a disulfide-stabilized variable domain (without a peptide linker) is fused to the C-terminus of the CH3 domain of an IgG.

[0325] Duobody

[0432] "Duobody" technology for producing bispecific antibodies with precise heavy chain pairing is known. DuoBody technology involves three basic steps to generate stable bispecific human IgG1 antibodies in a post-production exchange reaction. In the first step, two IgG1s, each containing a single matching mutation in the third constant (CH3) domain, are produced separately using a standard mammalian recombinant cell line. These IgG1 antibodies are then purified according to standard recovery and purification processes. After production and purification (post-production), the two antibodies are recombined under adapted experimental conditions, resulting in a bispecific antibody product with very high yields (typically >95%) (see, for example, Labrijn et al., PNAS 2013;110(13):5145-5150 and Labrijn et al. Nature Protocols 2014;9(10):2450-63, the contents of each of which are incorporated herein by reference).

[0326] Electrostatic interactions

[0433] A method for producing multifunctional antibodies has been disclosed in which CH3 amino acid changes are used with charged amino acids to make homodimer formation electrostatically unfavorable. EP 1870459 and WO 2009089004 describe other strategies that favor heterodimerization when different antibody domains are co-expressed in host cells. In these methods, one or more residues that constitute the heavy chain constant domain 3 (CH3) of both CH3 domains, the CH3-CH3 interface, are replaced with charged amino acids so that homodimer formation is electrostatically unfavorable and heterodimer formation is electrostatically favorable. Additional methods for creating multifunctional molecules using electrostatic interactions are described in the following references, the contents of each of which are incorporated herein by reference: U.S. Patent Application Publication No. 20100015133, U.S. Patent No. 8,592,562 B2, U.S. Patent No. 9,200,060 B2, U.S. Patent Application Publication No. 20140154254 A1, and U.S. Patent No. 9,358,286 A1.

[0327] Common light chain

[0434] Light chain mispairing must be avoided to produce a homogeneous preparation of bispecific IgG. One way to achieve this is to use the common light chain principle, i.e., to combine two binders that share one light chain but still have distinct specificities. An exemplary method for enhancing the formation of the desired bispecific antibody from a mixture of monomers is to provide a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions and methods for producing bispecific antibodies with a common light chain are disclosed, for example, in U.S. Pat. No. 7,183,076 B2, U.S. Patent Application Publication No. 20110177073 A1, European Patent No. 2,847,231 A1, International Publication No. 2016,079,081 A1, and European Patent No. 3,055,329 A1, the contents of each of which are incorporated herein by reference.

[0328] CrossMab

[0435] Another option for reducing light chain mispairing is the CrossMab technology, which avoids nonspecific light chain mispairing by exchanging the CH1 and CL domains in the Fab halves of a bispecific antibody. Such crossover variants retain binding specificity and affinity, but the two arms are different to prevent light chain mispairing. CrossMab technology (reviewed in Klein et al., supra) involves domain swapping between heavy and light chains to promote accurate pairing. Briefly, a two-step modification process is applied to construct bispecific IgG-like CrossMab antibodies capable of binding to two antigens using two distinct light chain-heavy chain pairs. First, a dimerization interface is engineered at the C-terminus of each heavy chain using a heterodimerization approach, such as knob-into-hole (KiH) technology, to ensure efficient formation of only two distinct heavy chain heterodimers from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B). Next, the constant heavy chain 1 (CH1) domain and the constant light chain (CL) domain of one antibody are exchanged (antibody A), while maintaining the variable heavy chain (VH) and variable light chain (VL) domains. The exchange of the CH1 and CL domains ensures that the modified antibody (antibody A) light chain dimerizes efficiently only with the modified antibody (antibody A) heavy chain, while the unmodified antibody (antibody B) light chain dimerizes efficiently only with the unmodified antibody (antibody B) heavy chain, thus ensuring that only the desired bispecific CrossMab is efficiently formed (see, for example, Cain, C. SciBX 4(28); doi:10.1038 / scibx.2011.783, the contents of which are incorporated herein by reference).

[0329] Common heavy chain

[0436] An exemplary method for enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain that interacts with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods for producing bispecific antibodies with a common heavy chain are disclosed, for example, in U.S. Patent Application Publication No. 20120184716, U.S. Patent Application Publication No. 20130317200, and U.S. Patent Application Publication No. 20160264685A1, the contents of each of which are incorporated herein by reference.

[0330] Amino acid modifications

[0437] Alternative compositions and methods for producing multifunctional antibodies with accurate light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers with one or more amino acid modifications in the CH1 and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or a combination thereof, which are part of the light and heavy chain interface, and create preferential pairing between each heavy chain and the desired light chain, such that when the two heavy chains and two light chains of a heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other (see, for example, WO2015181805). Other exemplary methods are described in International Publication No. 2016026943 (Argen-X), U.S. Patent Application Publication No. 20150211001, U.S. Patent Application Publication No. 20140072581A1, U.S. Patent Application Publication No. 20160039947A1, and U.S. Patent Application Publication No. 20150368352.

[0331] Lambda / Kappa format

[0438] Multifunctional molecules (e.g., multispecific antibody molecules) comprising lambda and kappa light chain polypeptides can be used to enable heterodimerization. Methods for producing bispecific antibody molecules comprising lambda and kappa light chain polypeptides are disclosed in PCT / US17 / 53053, filed September 22, 2017, designated International Publication No. WO 2018 / 057955, the entire contents of which are incorporated herein by reference.

[0332]

[0439] In some embodiments, the multifunctional molecule comprises a multispecific antibody molecule, e.g., an antibody molecule that comprises two binding specificities, e.g., a bispecific antibody molecule. A multispecific antibody molecule comprises: lambda light chain polypeptide 1 (LLCP1) specific for the first epitope; heavy chain polypeptide 1 (HCP1) specific for the first epitope; kappa light chain polypeptide 2 (KLCP2) specific for a second epitope; and Heavy chain polypeptide 2 (HCP2) specific for a second epitope Includes:

[0333]

[0440] "Lambda light chain polypeptide 1 (LLCP1)," as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence such that, when combined with a cognate heavy chain variable region, it can mediate specific binding to its epitope and form a complex with HCP1. In some embodiments, it includes all or a fragment of the CH1 region. In some embodiments, LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or a sequence thereof sufficient to mediate specific binding of its epitope and form a complex with HCP1. LLCP1, together with its HCP1, provides specificity for a first epitope (KLCP2, together with its HCP2, provides specificity for a second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than HCP2.

[0334]

[0441] "Kappa light chain polypeptide 2 (KLCP2)," as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence such that, when combined with a cognate heavy chain variable region, it is capable of mediating specific binding to its epitope and forming a complex with HCP2. In some embodiments, it comprises all or a fragment of the CH1 region. In some embodiments, KLCP2 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sufficient sequences thereof to mediate specific binding of its epitope and form a complex with HCP2. KLCP2, together with its HCP2, provides specificity for a second epitope (LLCP1, together with its HCP1, provides specificity for a first epitope).

[0335]

[0442] "Heavy chain polypeptide 1 (HCP1)," as that term is used herein, refers to a polypeptide that includes sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with cognate LLCP1, it is capable of mediating specific binding to its epitope and forming a complex with HCP1. In some embodiments, it includes all or a fragment of the CH1 region. In some embodiments, it includes all or a fragment of the CH2 and / or CH3 regions. In some embodiments, HCP1 includes HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences thereof sufficient to (i) mediate specific binding of its epitope and form a complex with LLCP1, (ii) preferentially form a complex with LLCP1 relative to KLCP2, as described herein; and (iii) preferentially form a complex with HCP2 relative to another molecule of HCP1, as described herein. HCP1, together with LLCP1, provides specificity for the first epitope (KLCP2, together with HCP2, provides specificity for the second epitope).

[0336]

[0443] "Heavy chain polypeptide 2 (HCP2)," as that term is used herein, refers to a polypeptide that includes sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with cognate LLCP1, it is capable of mediating specific binding to its epitope and forming a complex with HCP1. In some embodiments, it includes all or a fragment of the CH1 region. In some embodiments, it includes all or a fragment of the CH2 and / or CH3 regions. In some embodiments, HCP1 includes HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences sufficient to (1) mediate specific binding of its epitope and form a complex with KLCP2, (ii) preferentially form a complex with KLCP2 relative to LLCP1, as described herein; and (iii) preferentially form a complex with HCP1 relative to another molecule of HCP2, as described herein. HCP2, together with its KLCP2, provides specificity for the second epitope (LLCP1, together with its HCP1, provides specificity for the first epitope).

[0337]

[0444] In some embodiments, in the multifunctional polypeptide molecules described herein: LLCP1 has a higher affinity for HCP1 than for HCP2; and / or KLCP2 has a higher affinity for HCP2 than for HCP1.

[0338]

[0445] In some embodiments, the affinity of LLCP1 for HCP1 is sufficiently greater than its affinity for HCP2 such that under preselected conditions, e.g., in an aqueous buffer at pH 7, e.g., in saline at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multispecific antibody molecules have LLCP1 complexed or interfaced with HCP1.

[0339]

[0446] In some embodiments, in the multifunctional polypeptide molecules described herein: HCP1 has a higher affinity for HCP2 than for a second molecule of HCP1; and / or HCP2 has a higher affinity for HCP1 than for a second molecule of HCP2.

[0340]

[0447] In some embodiments, the affinity of HCP1 for HCP2 is sufficiently greater than the affinity of HCP1 for a second molecule such that under preselected conditions, e.g., in an aqueous buffer at pH 7, e.g., in saline at pH 7, or under physiological conditions, at least 75%, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multifunctional antibody molecules have HCP1 complexed or interfaced with HCP2.

[0341]

[0448] In another aspect, described herein is a method for generating or producing a multifunctional antibody molecule. The method comprises: (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three, or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both); (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three, or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both): (iii) providing a lambda chain polypeptide (e.g., a lambda light chain variable region (VLλ), a lambda light chain constant chain (VLλ), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH); and (iv) providing a kappa chain polypeptide (e.g., a lambda light chain variable region (VLλ), a lambda light chain constant chain (VLλ), or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH) under conditions in which (i) to (iv) associate; Includes:

[0342]

[0449] In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization.

[0450] In some embodiments, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In some embodiments, (i)-(iv) are expressed in the cell. In some embodiments, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into different cells, e.g., different mammalian cells, e.g., two or more CHO cells. In some embodiments, (i)-(iv) are expressed in the cell.

[0343]

[0451] In some embodiments, the method further comprises purifying the cell-expressed antibody molecules, eg, using lambda and / or kappa-specific purification, eg, affinity chromatography.

[0344]

[0452] In some embodiments, the method further comprises evaluating the cell-expressed multifunctional antibody molecule. For example, the purified cell-expressed multifunctional antibody molecule can be analyzed by techniques known in the art, including mass spectrometry. In some embodiments, the purified cell-expressed antibody molecule is cleaved, for example, digested with papain to generate Fab portions, and evaluated using mass spectrometry.

[0345]

[0453] In some embodiments, the methods produce correctly paired kappa / lambda multispecific, e.g., bispecific, antibody molecules in high yield, e.g., at least 75%, 80, 90, 95, 98, 99, 99.5, or 99.9% yield.

[0346]

[0454] In other embodiments, the multispecific, e.g., bispecific, antibody molecule comprises: (i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising one, two, three, or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)), e.g., the first heavy chain polypeptide in which the HCP1 binds to a first epitope; (ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising one, two, three, or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)), e.g., where the HCP2 binds a second epitope; (iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light chain variable region (VLλ), a lambda light chain constant chain (VLλ), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), e.g., the LLCP1 binds to a first epitope; and (iv) a kappa light chain polypeptide (KLCP2) (e.g., a kappa light chain variable region (VLκ), a kappa light chain constant chain (VLκ), or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH), e.g., where KLCP2 binds to a second epitope. Includes:

[0347]

[0455] In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization. In some embodiments, the multifunctional antibody molecule has a first binding specificity comprising a hybrid VLλ-CLλ heterodimerized to a first heavy chain variable region connected to Fc constant CH2-CH3 domains (with knob modifications), and a second binding specificity comprising a hybrid VLκ-CLκ heterodimerized to a second heavy chain variable region connected to Fc constant CH2-CH3 domains (with hole modifications). Multispecific or multifunctional antibody molecules

[0456] Exemplary structures of the multispecific and multifunctional molecules defined herein are described throughout. Exemplary structures are further described in Weidle U et al. (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10:1-18 (2013); and Spiess C et al. (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology 67:95-106), the entire contents of each of which are incorporated herein by reference.

[0348]

[0457] In some embodiments, a multispecific antibody molecule may contain more than one antigen-binding site, with different sites specific for different antigens. In some embodiments, a multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In some embodiments, a multispecific antibody molecule contains an antigen-binding site specific for a target cell (e.g., a cancer cell) and a different antigen-binding site specific for an immune effector cell. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with an additional antigen-binding moiety appended; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0349]

[0458] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two in one), DAF (four in one), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab. See Spiess et al. Mol. Immunol. 67 (2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, the BsIgG comprises a heavy chain engineered to heterodimerize. For example, heavy chains can be engineered to heterodimerize using a "knob-into-hole" strategy, the SEED platform, a common heavy chain (e.g., in κλ-bodies), and heterodimeric Fc regions. See Spiess et al. Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include duobodies, azymetrics, charge pairs, HA-TF, SEEDbodies, and differential Protein A affinity. See ibid. BsIgG can be produced by the separate expression of component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by the expression of component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., Protein A and sequential pH elution.

[0350]

[0459] IgG with additional antigen-binding moieties added is another format of bispecific antibody molecule. For example, monospecific IgG can be engineered to have bispecificity by adding additional antigen-binding units to the monospecific IgG, for example, to the N-terminus or C-terminus of either the heavy chain or the light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See ibid. Examples of added IgG formats include dual variab...

Claims

1. 1. A method of treating cancer in a human subject in need thereof, comprising administering to the human subject a multifunctional molecule, the multifunctional molecule comprising: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, The multifunctional molecule is administered to the human subject at a first dose of about 0.001 mg / kg to about 10 mg / kg, thereby treating cancer in the human subject.

2. 1. A method of treating cancer in a human subject in need thereof, comprising administering to the human subject a multifunctional molecule, the multifunctional molecule comprising: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, The method, wherein the administering step comprises administering multiple doses of the multifunctional molecule to the human subject.

3. 1. A method of treating cancer in a human subject in need thereof, comprising administering to the human subject a first dose of a multifunctional molecule, wherein the multifunctional molecule comprises: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, the human subject is characterized by having a solid tumor; and (i) if the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously been treated for the symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or more, is not currently receiving treatment for a CNS disease, and does not currently have leptomeningeal disease or spinal cord compression; and (ii) if the human subject has previously been treated with checkpoint inhibitor therapy (CPI), the human subject has remission of CPI immune-related toxicity to either Grade < 1 or baseline compared to before treatment with the CPI.

4. 1. A method of treating cancer in a human subject in need thereof, comprising administering to the human subject a first dose of a multifunctional molecule, wherein the multifunctional molecule comprises: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof wherein the human subject comprises (i) no history of autoimmune disease; (ii) has not undergone major surgery or has a traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject has no unhealed wounds from surgery or injury; (iii) not been treated with >10 mg / day of an immunosuppressant within 7 days prior to the first administration of the multifunctional molecule; (iv) has not been previously treated with cytotoxic chemotherapy, small molecule inhibitors, radiation therapy, or interventional radiology procedures within two weeks prior to the first administration of the multifunctional molecule; (v) have not been previously treated with a monoclonal antibody, antibody-drug conjugate, or radioimmunoconjugate within six weeks prior to the first administration of the multifunctional molecule; (vi) not having an inflammatory process that has not resolved within four weeks prior to the first administration of the multifunctional molecule; (vii) no clinically significant pulmonary dysfunction; or (viii) not having an active viral, bacterial, or systemic fungal infection requiring parenteral treatment within 7 days of the first administration of said multifunctional molecule.

5. 5. The method of claim 1, 3 or 4, wherein the first dose is the first of multiple doses.

6. 10. The method of claim 1, 2, or 4, wherein the human subject is characterized by having a solid tumor.

7. (i) if the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously been treated for symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or more, is not currently receiving treatment for a CNS disease, and does not currently have leptomeningeal disease or spinal cord compression; and (ii) if the human subject has previously been treated with checkpoint inhibitor therapy (CPI), the human subject has remission of CPI immune-related toxicity to either Grade < 1 or baseline compared to before treatment with the CPI.

8. 8. The method of any one of claims 3 or 5-7, wherein the solid tumor is selected from the group consisting of high mutational burden (TMB-H), microsatellite instability / DNA mismatch repair (MSI-H / dMMR), virus-associated tumors, metastatic triple-negative breast cancer (mTNBC), recurrent and refractory epithelial ovarian cancer, metastatic castration-resistant prostate cancer (mCRPC); K-Ras wild-type CRC; K-Ras mutant CRC, and primary stage IV or recurrent non-small cell lung cancer (NSCLC).

9. 9. The method of claim 8, wherein the virus-associated tumor comprises Merkel cell carcinoma, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

10. 8. The method of any one of claims 3 or 5-7, wherein the human subject is not concurrently being treated for a CNS disease, the subject does not have a leptomeningeal disease, or the subject does not have spinal cord compression.

11. 8. The method of any one of claims 3 or 5-7, wherein the subject's CPI immune-related toxicity is Grade ≦1 or baseline, and the subject is not experiencing CPI-associated endocrinopathy, or the subject is not experiencing CPI-associated Grade 3-4 pneumonitis, pericarditis / myocarditis, colitis and intestinal perforation, myositis, encephalitis, or peripheral neuropathy.

12. the human subject (i) No history of autoimmune disease other than vitiligo; psoriasis, atopic dermatitis, or other autoimmune skin conditions not requiring systemic treatment; Graves' disease that has been euthyroid for >4 weeks; hypothyroidism controlled with thyroid hormone replacement therapy; hair loss; arthritis that is managed without systemic treatment other than oral nonsteroidal anti-inflammatory drugs, and adrenal insufficiency that is well controlled with replacement therapy; (ii) has not undergone major surgery or has a traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject has no unhealed wounds from surgery or injury; (iii) not been treated with >10 mg / day of an immunosuppressant within 7 days prior to the first administration of the multifunctional molecule; (iv) has not been previously treated with cytotoxic chemotherapy, small molecule inhibitors, radiation therapy, or interventional radiology procedures within two weeks prior to the first administration of the multifunctional molecule; (v) have not been previously treated with a monoclonal antibody, antibody-drug conjugate, or radioimmunoconjugate within six weeks prior to the first administration of the multifunctional molecule; (vi) not having an inflammatory process that has not resolved within four weeks prior to the first administration of the multifunctional molecule; (vii) no clinically significant pulmonary dysfunction; or (viii) The method of claim 1, 2, 3, or 5, wherein the patient does not have an active viral infection, bacterial infection, or systemic fungal infection requiring parenteral treatment within 7 days of the first administration of the multifunctional molecule.

13. 13. The method of claim 4, 5, or 12, wherein the autoimmune disease does not include vitiligo, psoriasis, atopic dermatitis, or other autoimmune skin conditions that do not require systemic treatment; Graves' disease that has been euthyroid for >4 weeks now; hypothyroidism that is controlled with thyroid hormone replacement therapy; hair loss; arthritis that is managed without systemic treatment other than oral nonsteroidal anti-inflammatory drugs, and adrenal insufficiency that is well controlled with replacement therapy.

14. The method of any one of claims 1 to 13, wherein the human subject is at least 18 years old.

15. 15. The method of any one of claims 2 to 14, wherein the multifunctional molecule is administered to the human subject at a first dose of about 0.001 mg / kg to about 10 mg / kg.

16. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.001 mg / kg to about 1 mg / kg.

17. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.001 mg / kg to about 5 mg / kg.

18. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.001 mg / kg to about 10 mg / kg.

19. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.005 mg / kg to about 1 mg / kg.

20. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.005 mg / kg to about 5 mg / kg.

21. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.005 mg / kg to about 10 mg / kg.

22. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.01 mg / kg to about 1 mg / kg.

23. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.01 mg / kg to about 5 mg / kg.

24. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.01 mg / kg to about 10 mg / kg.

25. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.05 mg / kg to about 1 mg / kg.

26. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.05 mg / kg to about 5 mg / kg.

27. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.05 mg / kg to about 10 mg / kg.

28. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.1 mg / kg to about 1 mg / kg.

29. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.1 mg / kg to about 5 mg / kg.

30. 16. The method of claim 1 or 15, wherein the multifunctional molecule is administered in a first dose of about 0.1 mg / kg to about 10 mg / kg.

31. The multifunctional molecule is 0.001 mg / kg, 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.2 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.3 mg / kg, 0.31 mg / kg, 0.3^2 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.4 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.5 mg / kg, 0.51 mg / kg, 0.52 mg / kg, 0.53 mg / kg, {0.54 mg / kg, 0.55 mg / kg, 0.56 mg / kg, 0.57 mg / kg, 0.58 mg / kg, 0.59 mg / kg, 0.6 mg / kg, 0.61 mg / kg, 0.62 mg / kg, 0.63 mg / kg, 0.64 mg / kg, 0.65 mg / kg, 0.66 mg / kg, 0.67 mg / kg, 0.68 mg / kg, 0.69 mg / kg, 0.7 mg / kg, 0.71 mg / kg, 0.72 mg / kg, 0.73 mg / kg, 0.74 mg / kg, 0.75 mg / kg, 0.76 mg / kg, 0.77 mg / kg, 0.78 mg / kg, 0.79 mg / kg, 0.8 mg / kg, 0.81 mg / kg, 0.82 mg / kg, 0.83 mg / kg, 0.84 mg / kg, 0.85 mg / kg, 0.86 mg / kg, 0.87 mg / kg, 0.88 mg / kg, 0.89 mg / kg, 0.9 mg / kg, It should be noted that there seems to be a formatting issue in the original text where "0.3^2 mg / kg" might be incorrect. I've translated it as is, but it might need to be verified in the source context. Also, some parts like "{0.54 mg / kg, 0.55 mg / kg, 0.56 mg / kg, 0.57 mg / kg, 0.58 mg / kg, 0.59 mg / kg, 0.6 mg / kg, 0.61 mg / kg, 0.62 mg / kg, 0.63 mg / kg, 0.6^4 mg / kg, 0.65 mg / kg, 0.66 mg / kg, 0.67 mg / kg, 0.68 mg / kg, 0.69 mg / kg, 0.7 mg / kg, 0.71 mg / kg, 0.72 mg / kg, 0.73 mg / kg, 0.74 mg / kg, 0.75 mg / kg, 0.76 mg / kg, 0.77 mg / kg, 0.78 mg / kg, 0.79 mg / kg, 0.8 mg / kg, 0.81 mg / kg, 0.82 mg / kg, 0.83 mg / kg, 0.84 mg / kg, 0.85 mg / kg, 0.86 mg / kg, 0.87 mg / kg, 0.88 mg / kg, 0.89 mg / kg, 0.9 mg / kg," have some potential errors in formatting which are translated as accurately as possible while maintaining the original structure.01010、002000、00300 100000、005000、00000000000000000000000000 9990、1999.11 1500、2000、2500、3 4.

5.

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4. 5 050、550、650、550、 750、850、850 、95、955、105、105 11. 11.15.1.1.1.1.1.1.1.1 25000、13000、130000、14 1450、1550、1550 、1000、16500、1700、175 1000、1850、1900 、1955、255、2055、2 205、2155、225、225 230、2350、2450、24 55、255、2555、26 2050、2750、2750、2 85、2855、295、295 3.

35.

35. 3.5.3 15000、32000、320000、33 350、350、345 、30000、355000、30000、365 100、3000、30000、30000、 38500、30000、30500、40 4050、415、4150 、4200、42500、4300、43 55、445、4455、454 3. 4555.

465. 465 100、4750、485、485 1000、40000、または500 The 15th of the 15th of the 15th century.

32. 32. The method of any one of claims 1 and 3-31, wherein the administering step comprises administering multiple doses of the multifunctional molecule to the human subject.

33. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is a lower dose than a previous dose immediately preceding the subsequent dose and is administered after administration of the previous dose has been shown to be intolerable.

34. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is the same dose as a previous dose immediately preceding the subsequent dose, and is administered after administration of the previous dose has been shown to be tolerable.

35. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is a higher dose than a previous dose immediately preceding the subsequent dose and is administered after administration of the previous dose has been shown to be tolerable.

36. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is the same dose as a previous dose immediately preceding the subsequent dose, and is administered after administration of the previous dose has been shown to be effective.

37. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is a lower dose than a previous dose immediately preceding the subsequent dose and is administered after administration of the previous dose has been shown to be effective.

38. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is a higher dose than a previous dose immediately preceding the subsequent dose and is administered after administration of the previous dose has been shown to be ineffective.

39. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the previous dose that immediately precedes the subsequent dose.

40. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is administered at least 1, 2, 3, or 4 weeks after administration of the preceding dose that immediately precedes the subsequent dose.

41. 33. The method of claim 2 or 32, wherein a subsequent dose of the multiple doses is administered at least 1, 2, 3, 4, 5, 10, 11, or 12 months after administration of the preceding dose that immediately precedes the subsequent dose.

42. 33. The method of claim 2 or 32, wherein the frequency of administration of the multiple doses is maintained or reduced after the previous dose immediately preceding the subsequent dose is shown to be effective.

43. 33. The method of claim 2 or 32, wherein the administration frequency of the administering step is increased after one dose in the multiple doses is shown to be ineffective.

44. 44. The method of any one of claims 1 to 43, comprising administering the multifunctional molecule to the human subject once a week.

45. 45. The method of claim 44, comprising administering the multifunctional molecule to the human subject once a week for at least 1, 2, 3, or 4 weeks, or for at least 1, 2, 3, 4, 5, 10, 11, or 12 months, or for at least 1, 2, or 3 years.

46. 44. The method of any one of claims 1 to 43, comprising administering the multifunctional molecule to the human subject once every two weeks.

47. 47. The method of claim 46, comprising administering the multifunctional molecule to the human subject once every two weeks for at least 1, 2, 3, or 4 weeks, or for at least 1, 2, 3, 4, 5, 10, 11, or 12 months, or for at least 1, 2, or 3 years.

48. 44. The method of any one of claims 1 to 43, comprising administering the multifunctional molecule to the human subject once every three weeks.

49. 49. The method of claim 48, comprising administering the multifunctional molecule to the human subject once every three weeks for at least 1, 2, 3, or 4 weeks, or for at least 1, 2, 3, 4, 5, 10, 11, or 12 months, or for at least 1, 2, or 3 years.

50. 44. The method of any one of claims 1 to 43, wherein the multifunctional molecule is administered to the human subject once every two weeks for 28 days, during which the multifunctional molecule is administered to the human subject on days 1 and 15.

51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered by intravenous infusion.

52. 51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered subcutaneously, intratumorally, intranodally, intramuscularly, intradermally, or intraperitoneally.

53. 51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered by intravenous infusion over a time course of about 25 minutes to about 240 minutes.

54. 51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered by intravenous infusion over a time course of about 105 to 120 minutes or about 125 to 145 minutes.

55. 51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered by intravenous infusion over a time course of about 150 to 200 minutes or about 160 to 190 minutes.

56. 51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered by intravenous infusion over a time course of about 25 minutes to about 35 minutes or about 55 minutes to about 65 minutes.

57. 51. The method of any one of claims 1 to 50, wherein the multifunctional molecule is administered by intravenous infusion over a time course of about 35 minutes to about 50 minutes or about 85 minutes to about 95 minutes.

58. 58. The method of any one of claims 1 to 57, further comprising administering at least one additional therapeutic agent or treatment.

59. 59. The method of claim 58, wherein the at least one additional therapeutic agent or treatment is administered simultaneously with the dose of the multifunctional molecule.

60. 59. The method of claim 58, wherein the at least one additional therapeutic agent or treatment is administered prior to administration of the dose of the multifunctional molecule.

61. 59. The method of claim 58, wherein the at least one additional therapeutic agent or treatment is administered after administration of the dose of the multifunctional molecule.

62. 62. The method of any one of claims 1-61, wherein the administering step comprises administering a pharmaceutical composition comprising the multifunctional molecule, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

63. 63. The method of claim 62, wherein the pharmaceutical composition is a liquid composition.

64. 63. The method of claim 62, wherein the pharmaceutical composition comprises a pharmaceutically acceptable diluent that is saline.

65. 63. The method of claim 62, wherein the pharmaceutical composition comprises a pharmaceutically acceptable diluent that is 0.9% saline.

66. 66. The method of any one of claims 62-65, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL.

67. 67. The method of claim 66, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.2 mg / mL to about 15 mg / mL.

68. 67. The method of claim 66, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 1.5 mg / mL.

69. 67. The method of claim 66, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.2 mg / mL to about 1.5 mg / mL.

70. The multifunctional molecule is present in the pharmaceutical composition at about 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.30 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.40 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, 0.50 mg / mL, 0.51 mg / mL, 0.52 mg / mL, 0.53 mg / mL, 0.54 mg / mL, 0.55 mg / mL, 0.56 mg / mL, 0.57 mg / mL, 0.58 mg / mL, 0.59 mg / mL, 0.60 mg / mL, 0.61 mg .. 3mg / mL, 0.35mg / mL, 0.4mg / mL, 0.45mg / mL, 0.5mg / mL, 0.55mg / mL, 0.6mg / mL, 0.65mg / mL, 0.7mg / mL, 0.75mg / mL, 0.8mg / mL, 0.85mg / mL, 0.9mg / mL, 0.95mg / mL, 1mg / mL, 1.05mg / mL, 1.1mg / mL, 1.15mg / mL, 1.2mg / mL, 1.25mg / mL, 1.3mg / mL, 1.35mg / mL, 1.4mg / mL, 1.4 5mg / mL, 1.5mg / mL, 1.55mg / mL, 1.6mg / mL, 1.65mg / mL, 1.7mg / mL, 1.75mg / mL, 1.8mg / mL, 1.85mg / mL, 1.9mg / mL, 1.95mg / mL, 2mg / mL, 2.05mg / mL, 2.1mg / mL, 2.15mg / mL, 2.2mg / mL, 2.25mg / mL, 2.3mg / mL, 2.35mg / mL, 2.4mg / mL, 2.45mg / mL, 2.5mg / mL, 3mg / mL, 3.5mg / m 67. The method of claim 66, wherein the hydroxybenzoate is present at a concentration of 1000 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL, 13 mg / mL, 13.5 mg / mL, 14 mg / mL, 14.5 mg / mL, or 15 mg / mL.

71. 71. The method of any one of claims 62-70, wherein the pharmaceutical composition comprises from about 0.5 mL to about 500 mL of diluent.

72. A dose of a pharmaceutical composition comprising a multifunctional molecule, The multifunctional molecule is (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, The dose is from about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule.

73. 73. The dose of claim 72, wherein the dose is from about 0.001 mg / kg to about 1 mg / kg of the multifunctional molecule.

74. 73. The dose of claim 72, wherein the dose is from about 0.001 mg / kg to about 5 mg / kg of the multifunctional molecule.

75. 73. The dose of claim 72, wherein the dose is from about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule.

76. 73. The dose of claim 72, wherein the dose is from about 0.005 mg / kg to about 1 mg / kg of the multifunctional molecule.

77. 73. The dose of claim 72, wherein the multifunctional molecule is administered in a first dose of about 0.005 mg / kg to about 5 mg / kg of the multifunctional molecule.

78. 73. The dose of claim 72, wherein the dose is from about 0.005 mg / kg to about 10 mg / kg of the multifunctional molecule.

79. 73. The dose of claim 72, wherein the dose is from about 0.01 mg / kg to about 1 mg / kg of the multifunctional molecule.

80. 73. The dose of claim 72, wherein the dose is from about 0.01 mg / kg to about 5 mg / kg of the multifunctional molecule.

81. 73. The dose of claim 72, wherein the dose is from about 0.01 mg / kg to about 10 mg / kg of the multifunctional molecule.

82. 73. The dose of claim 72, wherein the dose is from about 0.05 mg / kg to about 1 mg / kg of the multifunctional molecule.

83. 73. The dose of claim 72, wherein the dose is from about 0.05 mg / kg to about 5 mg / kg of the multifunctional molecule.

84. 73. The dose of claim 72, wherein the dose is from about 0.05 mg / kg to about 10 mg / kg of the multifunctional molecule.

85. 73. The dose of claim 72, wherein the dose is from about 0.1 mg / kg to about 1 mg / kg of the multifunctional molecule.

86. 73. The dose of claim 72, wherein the dose is from about 0.1 mg / kg to about 5 mg / kg of the multifunctional molecule.

87. 73. The dose of claim 72, wherein the dose is from about 0.1 mg / kg to about 10 mg / kg of the multifunctional molecule.

88. Approximately 0.001 mg / kg, 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.2 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.3 mg / kg, 0.31 mg / kg, 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.4 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.5 mg / kg, 0.51 mg / kg, 0.52 mg / kg, 0.53 mg / kg, 0.54 mg / kg, 0.55 mg / kg, 0.56 mg / kg, 0.57 mg / kg, 0.58 mg / kg, 0.59 mg / kg, 0.6 mg / kg, 0.61 mg / kg, 0.62 mg / kg, 0.63 mg / kg, 0.64 mg / kg, 0.65 mg / kg, 0.66 mg / kg, 0.67 mg / kg, 0.68 mg / kg, 0.69 mg / kg, 0.7 mg / kg, 0.71 mg / kg, 0.72 mg / kg, 0.73 mg / kg, 0.74 mg / kg, 0.75 mg / kg, 0.76 mg / kg, 0.77 mg / kg, 0.78 mg / kg, 0.79 mg / kg, 0.8 mg / kg, 0.81 mg / kg, 0.82 mg / kg, 0.83 mg / kg, 0.84 mg / kg, 0.85 mg / kg, 0.86 mg / kg, 0.87 mg / kg, 0.88 mg / kg, 0.89 mg / kg, 0.9 mg / kg, 0.91 mg / kg,FA2000、003000、004000、 09500、00600、000000 999999991115 10、2000、25000、3000、3 4.

15.

45. 4.5.5 5000、Footnote、Footnote、Footnote、 7500、8000、8500、Nine 、9500、1050、10500、1 1000、11500、12000、1250 4.

13.

13.

14. 14 550、1555、1555、16 1650、1650、1750 、1850、1850、19th、19 50、2000、20500、210 12.25.2.25.2.2.5.2 1000、23500、2400、245 250、2550、260、 2050、2050、2450、28 2859 2959 2955 30.

35.

35. 3.5 5000、32000、300000 350、350、345 、30000、355000、30000、365 100、3000、30000、3000 4. 35000、30000、39500 100、40500、4100、415 12、425、4255、430、 4550、4450、4455 4556、465、465 48. 475.48.48 50.

49.

49. 4.5.5 Thank you for watching this video, please note 72.

89. 1. A pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, A pharmaceutical composition wherein the pharmaceutically acceptable diluent is saline.

90. 90. The pharmaceutical composition of claim 89, wherein the pharmaceutically acceptable diluent is 0.9% saline.

91. 91. The dose of any one of claims 69 to 88 or the pharmaceutical composition of claim 89 or 90, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL or about 0.2 mg / mL to about 1.5 mg / mL.

92. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 0.5 mL to about 500 mL.

93. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 5 mL to about 500 mL.

94. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 50 mL to about 500 mL.

95. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 0.5 mL to about 350 mL.

96. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 0.5 mL to about 250 mL.

97. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 0.5 mL to about 150 mL.

98. The dose of any one of claims 69 to 88 or the pharmaceutical composition of any one of claims 89 to 91, wherein the total volume of the pharmaceutical composition is from about 0.5 mL to about 50 mL.

99. 1. A pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, The pharmaceutical composition, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL.

100. 1. A pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, A pharmaceutical composition comprising about 0.1 mg to about 500 mg of the multifunctional molecule.

101. 101. The pharmaceutical composition of claim 99 or 100, comprising about 0.5 mg to about 200 mg of the multifunctional molecule.

102. 101. The pharmaceutical composition of claim 99 or 100, comprising about 0.5 mg to about 100 mg of the multifunctional molecule.

103. 101. The pharmaceutical composition of claim 99 or 100, comprising about 1 mg to about 200 mg of the multifunctional molecule.

104. 101. The pharmaceutical composition of claim 99 or 100, comprising about 1 mg to about 100 mg of the multifunctional molecule.

105. A pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable excipient, wherein the multifunctional molecule comprises: (a) a TCRβV6-binding portion, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof Including, A pharmaceutical composition, wherein the pharmaceutically acceptable excipient comprises one or more of L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

106. 106. The pharmaceutical composition of claim 105, comprising about 0.1 mg to about 500 mg of the multifunctional molecule.

107. 106. The pharmaceutical composition of claim 105, comprising about 0.5 mg to about 200 mg of the multifunctional molecule.

108. 106. The pharmaceutical composition of claim 105, comprising about 0.5 mg to about 100 mg of the multifunctional molecule.

109. 106. The pharmaceutical composition of claim 105, comprising about 1 mg to about 200 mg of the multifunctional molecule.

110. 106. The pharmaceutical composition of claim 105, comprising about 1 mg to about 100 mg of the multifunctional molecule.

111. 111. The pharmaceutical composition of any one of claims 105 to 110, comprising about 1 mM to about 200 mM, about 2 mM to about 100 mM, about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 20 mM L-histidine / L-histidine monohydrochloride buffer.

112. 112. The pharmaceutical composition of claim 105 or 111, comprising about 1% (wt / vol) to about 20% (wt / vol), about 2% (wt / vol) to about 15% (wt / vol), 5% (wt / vol) to about 12% (wt / vol), about 6% (wt / vol) to about 10% (wt / vol), about 8% (wt / vol) sucrose.

113. 113. The pharmaceutical composition of any one of claims 105-112, comprising about 0.001% (wt / vol) to about 0.1% (wt / vol), about 0.002% (wt / vol) to about 0.08% (wt / vol), 0.005% (wt / vol) to about 0.06% (wt / vol), about 0.008% (wt / vol) to about 0.04% (wt / vol), about 0.01% (wt / vol) to about 0.03% (wt / vol), about 0.02% (wt / vol) polysorbate-80.

114. 114. The pharmaceutical composition of any one of claims 105-113, comprising the multifunctional molecule at a concentration of about 0.5 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, about 2 mg / mL to about 80 mg / mL, about 4 mg / mL to about 50 mg / mL, about 6 mg / mL to about 20 mg / mL, about 8 mg / mL to about 12 mg / mL, or about 10 mg / mL.

115. 105. The dose of any one of claims 72 to 88, or the pharmaceutical composition of any one of claims 89 to 104, wherein the pharmaceutical composition comprises one or more of L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

116. the multifunctional molecule comprises a first polypeptide, a second polypeptide, and a third polypeptide; and the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous; (i) the first polypeptide comprises a first portion of a dimerization module linked to a first portion of the TCRβ V6 binding portion comprising the VH of the TCRβ V6 binding portion; (ii) the second polypeptide comprises a second portion of the dimerization module, and the IL-2, or a functional fragment or variant thereof, is covalently linked to the second polypeptide; and (iii) the third polypeptide comprises a second portion of the TCRβ V6 binding portion comprising the VL of the TCRβ V6 binding portion; A method according to any one of claims 1 to 71, a dose according to any one of claims 72 to 88, or a pharmaceutical composition according to any one of claims 89 to 115.

117. 117. The method, dose, or pharmaceutical composition of claim 116, wherein the first polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3517, 4000, 4004, 4006, 4008, 4010, 4011, 4014, 4016, and 4018; the second polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3521, 4002, 4007, 4003, 4013, and 4015; and the third polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3518, 4005, 4009, 4012, and 4017.

118. the multifunctional molecule comprises a first polypeptide and a second polypeptide; the first polypeptide and the second polypeptide are discontinuous, and the TCRβV6 binding portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single domain antibody; (i) the first polypeptide comprises a first portion of a dimerization module linked to the TCRβV6 binding portion; and (ii) the second polypeptide comprises a second portion of the dimerization module, and the IL-2, or a functional fragment or variant thereof, is covalently linked to the second polypeptide; A method according to any one of claims 1 to 71, a dose according to any one of claims 72 to 88, or a pharmaceutical composition according to any one of claims 89 to 115.

119. 119. The method, dose, or pharmaceutical composition of claim 118, wherein the first polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4019, 4021, 4023, 4025, and 4027, and the second polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4020, 4022, 4024, 4026, and 4028.

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