Multifunctional molecule combined with TCR and application thereof

Abstract

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

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 379,271, filed Oct. 12, 2022, and U.S. Provisional Application No. 63 / 381,231, filed Oct. 27, 2022, each of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Currently available molecules designed to redirect T cells for cancer immunotherapy to promote tumor cell lysis generally target the CD3ε (CD3e) subunit of the T cell receptor (TCR). However, this approach has limitations. Previous studies have shown, for example, that low doses of anti-CD3e monoclonal antibodies (mAbs) can cause T cell dysfunction and exert an immunosuppressive effect. In addition, anti-CD3e mAbs bind to all T cells, thereby activating a large number of T cells. This non-physiologically large-scale activation of T cells by these anti-CD3e mAbs may lead to the production of pro-inflammatory cytokines such as IFN-γ, IL-1-β, IL-6, IL-10, and TNF-α, resulting in a "cytokine storm", called cytokine release syndrome (CRS), which is also associated with neurotoxicity (NT). Accordingly, there is a need for improved T cell receptor-binding molecules that redirect T cells for cancer immunotherapy. SUMMARY OF THE INVENTION

[0004] Provided herein is a method of treating cancer in a human subject in need thereof, the method comprising 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, wherein the multifunctional molecule is administered to the human subject at a first dose of from about 0.001 mg / kg to about 10 mg / kg; thereby treating cancer in the human subject.

[0005] Also provided herein is a method of treating cancer in a human subject in need thereof, the method comprising 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, wherein the administering comprises administering multiple doses of the multifunctional molecule to the human subject.

[0006] The present invention also provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject a first dose of a multifunctional molecule, wherein the multifunctional molecule comprises a TCRβV6 binding moiety and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, wherein the human subject is characterized as having a solid tumor, and wherein if the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously received treatment for the symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or longer, and is not currently receiving treatment for CNS disease and does not currently have leptomeningeal disease or spinal cord compression; and if the human subject has previously received checkpoint inhibitor therapy (CPI), the CPI immune-related toxicity of the human subject has resolved to ≤ grade 1 or relative to the baseline prior to receiving CPI therapy.

[0007] The present invention also provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject a first dose of a multifunctional molecule, wherein the multifunctional molecule comprises a TCRβV6 binding moiety 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 had major surgery or traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject has no unhealed wound from surgery or injury; has not received immunosuppressive drugs at a daily dose > 10 mg within 7 days prior to the first administration of the multifunctional molecule; has not previously received cytotoxic chemotherapy, small molecule inhibitors, radiotherapy, or interventional radiology procedures within 2 weeks prior to the first administration of the multifunctional molecule; has not previously received monoclonal antibodies, antibody-drug conjugates, radioimmunoconjugates within 6 weeks prior to the first administration of the multifunctional molecule; has no unresolved inflammatory process within 4 weeks prior to the first administration of the multifunctional molecule; has no clinically significant pulmonary impairment; or has no active viral, bacterial, or systemic fungal infection requiring parenteral treatment within 7 days of the first administration of the multifunctional molecule.

[0008] In some embodiments, the first dose is the first dose of a multi-dose regimen. In some embodiments, the human subject is characterized as having a solid tumor.

[0009] In some embodiments, if the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously received treatment for the symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or longer, and is not currently receiving treatment for CNS disease and does not currently have leptomeningeal disease or spinal cord compression; and if the human subject has previously received checkpoint inhibitor therapy (CPI), the CPI immune-related toxicity of the human subject has resolved to ≤ grade 1 or relative to the baseline prior to receiving CPI therapy.

[0010] In some embodiments, the solid tumor is selected from high tumor 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).

[0011] In some embodiments, the virus-associated tumors include Merkel cell carcinoma, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

[0012] In some embodiments, the human subject is not receiving treatment for CNS diseases concurrently, the subject does not have leptomeningeal disease, or the subject does not have spinal cord compression.

[0013] In some embodiments, the CPI immune-related toxicity of the subject is ≤ grade 1 or at baseline, the subject has experienced CPI-related endocrine abnormalities, or the subject has not experienced CPI-related grade 3-4 pneumonia, pericarditis / myocarditis, colitis and intestinal perforation, myositis, encephalitis, or peripheral neuropathy.

[0014] In some embodiments, the human subject: has no history of autoimmune diseases other than the following: vitiligo; psoriasis, atopic dermatitis, or other autoimmune skin conditions that do not require systemic treatment; Graves' disease, with normal thyroid function for > 4 weeks; hypothyroidism controlled by thyroid replacement; alopecia; arthritis that can be controlled without systemic therapy other than oral non-steroidal anti-inflammatory drugs, and adrenal insufficiency well-controlled by replacement therapy; has not had major surgery or traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject does not have an unhealed wound from surgery or injury; has not received immunosuppressive drug treatment at a daily dose > 10 mg within 7 days prior to the first administration of the multifunctional molecule; has not previously received cytotoxic chemotherapy, small molecule inhibitors, radiotherapy, or interventional radiology procedures within 2 weeks prior to the first administration of the multifunctional molecule; has not previously received monoclonal antibodies, antibody-drug conjugates, or radioimmunoconjugates within 6 weeks prior to the first administration of the multifunctional molecule; has no unresolved inflammatory process within 4 weeks prior to the first administration of the multifunctional molecule; has no clinically significant pulmonary impairment; or has no active viral, bacterial, or systemic fungal infections requiring parenteral treatment within 7 days prior to the first administration of the multifunctional molecule.

[0015] 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, with normal thyroid function for > 4 weeks; hypothyroidism controlled by thyroid replacement; alopecia; arthritis that can be controlled without systemic therapy other than oral non-steroidal anti-inflammatory drugs, and adrenal insufficiency well controlled by replacement therapy.

[0016] In some embodiments, the human subject is at least 18 years old.

[0017] In some embodiments, the multifunctional molecule is administered to the 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.

[0018] 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, 0.2 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.Administered at a first dose of 9 mg / kg, 0.91 mg / kg, 0.92 mg / kg, 0.93 mg / kg, 0.94 mg / kg, 0.95 mg / kg, 0.96 mg / kg, 0.97 mg / kg, 0.98 mg / kg, 0.99 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, 20 mg / kg, 20.5 mg / kg, 21 mg / kg, 21.5 mg / kg, 22 mg / kg, 22.5 mg / kg, 23 mg / kg, 23.5 mg / kg, 24 mg / kg, 24.5 mg / kg, 25 mg / kg, 25.5 mg / kg, 26 mg / kg, 26.5 mg / kg, 27 mg / kg, 27.5 mg / kg, 28 mg / kg, 28.5 mg / kg, 29 mg / kg, 29.5 mg / kg, 30 mg / kg, 30.5 mg / kg, 31 mg / kg, 31.5 mg / kg, 32 mg / kg, 32.5 mg / kg, 33 mg / kg, 33.5 mg / kg, 34 mg / kg, 34.5 mg / kg, 35 mg / kg, 35.5 mg / kg, 36 mg / kg, 36.5 mg / kg, 37 mg / kg, 37.5 mg / kg, 38 mg / kg, 38.5 mg / kg, 39 mg / kg, 39.5 mg / kg, 40 mg / kg, 40.5 mg / kg, 41 mg / kg, 41.5 mg / kg, 42 mg / kg, 42.5 mg / kg, 43 mg / kg, 43.5 mg / kg, 44 mg / kg, 44.5 mg / kg, 45 mg / kg, 45.5 mg / kg, 46 mg / kg, 46.5 mg / kg, 47 mg / kg, 47.5 mg / kg, 48 mg / kg, 48.5 mg / kg, 49 mg / kg, 49.5 mg / kg or 50 mg / kg.

[0019] In some embodiments, administration comprises administering multiple doses of a multifunctional molecule to a human subject.

[0020] In some embodiments, following indication of intolerance to administration of a previous dose, a subsequent dose in the multiple doses is lower than the previous dose immediately preceding the subsequent dose. In some embodiments, following indication of tolerance to administration of a previous dose, a subsequent dose in the multiple doses is the same as the previous dose immediately preceding the subsequent dose. In some embodiments, following indication of tolerance to administration of a previous dose, a subsequent dose in the multiple doses is higher than the previous dose immediately preceding the subsequent dose.

[0021] In some embodiments, following indication of efficacy of a previous dose, a subsequent dose in the multiple doses is the same as the previous dose immediately preceding the subsequent dose. In some embodiments, following indication of efficacy of a previous dose, a subsequent dose in the multiple doses is lower than the previous dose immediately preceding the subsequent dose. In some embodiments, following indication of ineffectiveness of a previous dose, a subsequent dose in the multiple doses is higher than the previous dose immediately preceding the subsequent dose.

[0022] In some embodiments, a subsequent dose in 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 immediately preceding the subsequent dose. In some embodiments, a subsequent dose in the multiple doses is administered at least 1, 2, 3, or 4 weeks after administration of the previous dose immediately preceding the subsequent dose. In some embodiments, a subsequent dose in the multiple doses is administered at least 1, 2, 3, 4, 5, 10, 11, or 12 months after administration of the previous dose immediately preceding the subsequent dose.

[0023] In some embodiments, following indication of efficacy of the previous dose immediately preceding a subsequent dose, the dosing frequency of the multiple doses is maintained or decreased. In some embodiments, following indication of ineffectiveness of a dose in the multiple doses, the dosing frequency of administration is increased. In some embodiments, the method comprises administering the multifunctional molecule to the human subject once a week.

[0024] In some embodiments, the method comprises administering a multifunctional molecule to a human subject once a week 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 the human subject once every two weeks. In some embodiments, the method comprises administering the multifunctional molecule to the human subject once every two 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 the human subject once every three weeks. In some embodiments, the method comprises administering the multifunctional molecule to the 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 the human subject once every two weeks for 28 days, wherein the multifunctional molecule is administered to the human subject on day 1 and day 15.

[0025] 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.

[0026] In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of from about 25 minutes to about 240 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of from about 105 minutes to 120 minutes or from about 125 minutes to 145 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of from about 150 minutes to 200 minutes or from about 160 minutes to 190 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of from about 25 minutes to about 35 minutes or from about 55 minutes to about 65 minutes. In some embodiments, the multifunctional molecule is administered by intravenous infusion over a time course of from about 35 minutes to about 50 minutes or from about 85 minutes to about 95 minutes.

[0027] In some embodiments, the method further comprises administering at least one additional therapeutic agent or therapy. In some embodiments, the at least one additional therapeutic agent or therapy is administered concomitantly with a dose of the multifunctional molecule. In some embodiments, the at least one additional therapeutic agent or therapy is administered prior to a dose of the multifunctional molecule. In some embodiments, the at least one additional therapeutic agent or therapy is administered after a dose of the multifunctional molecule.

[0028] In some embodiments, administration comprises administering a pharmaceutical composition comprising a multifunctional molecule, and 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 a saline solution. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent that is a 0.9% saline solution.

[0029] 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.

[0030] In some embodiments, the multifunctional molecule is present in the pharmaceutical composition at a concentration of 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, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.05 mg / mL, 1.1 mg / mL, 1.15 mg / mL, 1.2 mg / mL, 1.25 mg / mL, 1.3 mg / mL, 1.35 mg / mL, 1.4 mg / mL, 1.45 mg / mL, 1.5 mg / mL, 1.55 mg / mL, 1.6 mg / mL, 1.65 mg / mL, 1.7 mg / mL, 1.75 mg / mL, 1.8 mg / mL, 1.85 mg / mL, 1.9 mg / mL, 1.95 mg / mL, 2 mg / mL, 2.05 mg / mL, 2.1 mg / mL, 2.15 mg / mL, 2.2 mg / mL, 2.25 mg / mL, 2.3 mg / mL, 2.35 mg / mL, 2.4 mg / mL, 2.45 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 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.

[0031] In some embodiments, the pharmaceutical composition comprises from about 0.5 mL to about 500 mL of diluent.

[0032] The present invention also provides a dosage of a pharmaceutical composition comprising a multifunctional molecule, wherein the multifunctional molecule comprises a TCRβV6 binding moiety and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the dosage is from about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule.

[0033] In some embodiments, the dosage is from about 0.001 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.001 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.005 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the multifunctional molecule is administered at a first dosage of from about 0.005 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.005 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.01 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.01 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.01 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.05 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.05 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.05 mg / kg to about 10 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.1 mg / kg to about 1 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.1 mg / kg to about 5 mg / kg of the multifunctional molecule. In some embodiments, the dosage is from about 0.1 mg / kg to about 10 mg / kg of the multifunctional molecule.

[0034] In some embodiments, the doses are about 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.Multifunctional molecules of 9 mg / kg, 0.91 mg / kg, 0.92 mg / kg, 0.93 mg / kg, 0.94 mg / kg, 0.95 mg / kg, 0.96 mg / kg, 0.97 mg / kg, 0.98 mg / kg, 0.99 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, 20 mg / kg, 20.5 mg / kg, 21 mg / kg, 21.5 mg / kg, 22 mg / kg, 22.5 mg / kg, 23 mg / kg, 23.5 mg / kg, 24 mg / kg, 24.5 mg / kg, 25 mg / kg, 25.5 mg / kg, 26 mg / kg, 26.5 mg / kg, 27 mg / kg, 27.5 mg / kg, 28 mg / kg, 28.5 mg / kg, 29 mg / kg, 29.5 mg / kg, 30 mg / kg, 30.5 mg / kg, 31 mg / kg, 31.5 mg / kg, 32 mg / kg, 32.5 mg / kg, 33 mg / kg, 33.5 mg / kg, 34 mg / kg, 34.5 mg / kg, 35 mg / kg, 35.5 mg / kg, 36 mg / kg, 36.5 mg / kg, 37 mg / kg, 37.5 mg / kg, 38 mg / kg, 38.5 mg / kg, 39 mg / kg, 39.5 mg / kg, 40 mg / kg, 40.5 mg / kg, 41 mg / kg, 41.5 mg / kg, 42 mg / kg, 42.5 mg / kg, 43 mg / kg, 43.5 mg / kg, 44 mg / kg, 44.5 mg / kg, 45 mg / kg, 45.5 mg / kg, 46 mg / kg, 46.5 mg / kg, 47 mg / kg, 47.5 mg / kg, 48 mg / kg, 48.5 mg / kg, 49 mg / kg, 49.5 mg / kg or 50 mg / kg.

[0035] The present invention also provides a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises a TCRβV6 binding moiety, and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the pharmaceutically acceptable diluent is a saline solution.

[0036] In some embodiments, the pharmaceutically acceptable diluent is a 0.9% saline solution.

[0037] 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.

[0038] The present invention also provides a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises a TCRβV6 binding moiety, and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of about 0.02 mg / mL to about 15 mg / mL.

[0039] The present invention also provides a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable diluent, wherein the multifunctional molecule comprises a TCRβV6 binding moiety, and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the pharmaceutical composition comprises about 0.1 mg to about 500 mg of the multifunctional molecule.

[0040] 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.

[0041] The present invention also provides a pharmaceutical composition comprising a multifunctional molecule and a pharmaceutically acceptable excipient, wherein the multifunctional molecule comprises: a TCRβV6 binding moiety, and interleukin-2 (IL-2) or a functional fragment or functional variant thereof, and wherein the pharmaceutically acceptable excipient comprises one or more of an L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

[0042] In some embodiments, the pharmaceutical composition comprises from about 0.1 mg to about 500 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises from about 0.5 mg to about 200 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises from about 0.5 mg to about 100 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises from about 1 mg to about 200 mg of the multifunctional molecule. In some embodiments, the pharmaceutical composition comprises from about 1 mg to about 100 mg of the multifunctional molecule.

[0043] In some embodiments, the pharmaceutical composition comprises from about 1 mM to about 200 mM, from about 2 mM to about 100 mM, from about 10 mM to about 50 mM, from about 15 mM to about 25 mM, or about 20 mM of the L-histidine / L-histidine monohydrochloride buffer.

[0044] In some embodiments, the pharmaceutical composition comprises from about 1% (w / v) to about 20% (w / v), from about 2% (w / v) to about 15% (w / v), 5% (w / v) to about 12% (w / v), from about 6% (w / v) to about 10% (w / v), about 8% (w / v) of sucrose.

[0045] In some embodiments, the pharmaceutical composition comprises from about 0.001% (w / v) to about 0.1% (w / v), from about 0.002% (w / v) to about 0.08% (w / v), 0.005% (w / v) to about 0.06% (w / v), from about 0.008% (w / v) to about 0.04% (w / v), from about 0.01% (w / v) to about 0.03% (w / v), about 0.02% (w / v) of polysorbate-80.

[0046] In some embodiments, the pharmaceutical composition comprises the multifunctional molecule at a concentration of from about 0.5 mg / mL to about 200 mg / mL, from about 1 mg / mL to about 100 mg / mL, from about 2 mg / mL to about 80 mg / mL, from about 4 mg / mL to about 50 mg / mL, from about 6 mg / mL to about 20 mg / mL, from about 8 mg / mL to about 12 mg / mL, or about 10 mg / mL.

[0047] In some embodiments, the pharmaceutical composition comprises one or more of an L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

[0048] In some embodiments, the multifunctional molecule comprises a first polypeptide, a second polypeptide, and a third polypeptide; wherein the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous, wherein the first polypeptide comprises a first portion of a dimerization module, the first portion of the dimerization module being linked to a first portion of a TCRβV6-binding portion, the first portion of the TCRβV6-binding portion comprising the VH of the TCRβV6-binding portion; the second polypeptide comprises a second portion of the dimerization module, wherein IL-2 or a functional fragment or functional variant thereof is covalently linked to the second polypeptide; and the third polypeptide comprises a second portion of the TCRβV6-binding portion, the second portion of the TCRβV6-binding portion comprising the VL of the TCRβV6-binding portion.

[0049] 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.

[0050] In some embodiments, the multifunctional molecule comprises a first polypeptide and a second polypeptide; wherein the first polypeptide and the second polypeptide are discontinuous, wherein the TCRβV6-binding portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), or a single domain antibody, wherein the first polypeptide comprises a first portion of a dimerization module linked to the TCRβV6-binding portion; and the second polypeptide comprises a second portion of the dimerization module, wherein IL-2 or a functional fragment or functional variant thereof is covalently linked to the second polypeptide.

[0051] 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.

[0052] Incorporated by reference

[0053] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The novel features of the present disclosure are particularly set forth 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 of illustrative embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings, in which:

[0055] Figure 1 An overview of the treatment and assessment plan for this study is shown.

[0056] Figure 2 Shown are the predicted effects on human Vβ6 / Vβ10 CD8+ T cell expansion (frequency) after a single 1-hour IV infusion of different dose levels of Compound 1. Body weight 70 kg. Solid line - median, shaded area - 5th and 95th percentiles of 1000 simulated individuals, dashed line - 20% increase from baseline.

[0057] Figure 3 Compound 1 pharmacology, MABEL estimates, and C values for Phase 1 dose escalation are shown. 最大 MABEL estimates 1: First diamond on the left: EC20 for in vitro human Vβ6 / V10 CD8+ T cell expansion (0.7 nM) and second diamond on the left: C20 at the modeled ED20 dose from the IV monkey study 最大 (0.91 nM). MABEL estimate 2: predicted human C associated with a starting dose of 0.04 mg / kg 最大 (7.98nM), at which starting dose ≥95% of patients are predicted to have a 20% increase in Vβ6 / V10 CD8+T cell expansion over baseline. * In a repeated-dose GLP toxicology study, moderate hunched posture, decreased activity, and decreased appetite were observed in monkeys dosed with 1 mg / kg IV Compound 1 4-5 days after infusion, and in a single-dose non-GLP pharmacology study, monkeys dosed with 1.5 mg / kg Compound 1 developed fever, diarrhea, and mild dehydration. These signs resolved by day 8-9 after dosing. In a repeated-dose non-GLP monkey pharmacology study, occasional and transient hunched posture and decreased activity were observed at the 0.5 mg / kg dose level.

[0058] Figure 4 A phase 1 trial design is shown.

[0059] Figure 5 It is shown 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.

[0060] Figure 6 Shows potent monotherapy activity of murine surrogate bispecific antibody (BsAb) of mSTAR, with durable responses in multiple tumor models including PD-1 refractory models.

[0061] Figure 7 Shows that mSTAR causes potent tumor regression in the EMT6 model.

[0062] Figures 8A-8B Shows that mSTAR remodels tumor-infiltrating lymphocytes (TILs), such as the expansion of 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.

[0063] Figure 9 Shows that mSTAR induces a novel TEM phenotype. For each violin plot, the vehicle is on the left and mSTAR is on the right.

[0064] Figures 10A-10B Shows that mSTAR induces an increase in TCR diversity in TILs. Figure 10A Shows that mSTAR increases Vβ TIL clonal diversity. Figure 10B Shows a substantial increase in unique CDR3 transcripts in TILs treated with mSTAR.

[0065] Figure 11 Shows that compound 1 induces the expansion of Vβ6CD8+ T cells in monkey blood with minimal Tregs.

[0066] Figure 12 Shows that compared to pembrolizumab, compound 1 induces ex vivo expansion of patient TILs and killing of refractory autologous tumors.

[0067] Figure 13 Shows that mSTAR promotes "functional memory" and long-term protection mediated by VβCD8+ T cells.

[0068] Figure 14 Shows that compared to isotype control, compound 1 and a multifunctional molecule containing a non-TCR binding domain and an IL-2 control increase IL-2R signaling (as measured by pSTAT5 levels).

[0069] Figure 15Shows the prevalence of Vβ6 TCR T cells in TIL and PBMC isolated from cancer patients (n = 43) and healthy donors (n = 20). For each cancer type, the bar on the left represents Vβ6-5 + TIL, and the bar on the right represents Vβ6-5 + PB.

[0070] Figure 16 Is a schematic diagram depicting an exemplary embodiment of a multifunctional molecule comprising a TCRβV binding moiety and a cytokine polypeptide (such as IL2) as described herein.

[0071] Figure 17 Shows that compound 1 binds to human CD4+ and CD8+ T cells similarly to a single-arm anti-Vβ6 / Vβ10 control.

[0072] Figure 18 Shows the IL-2 bioactivity as pSTAT5 activity among compound 1, RSV-IL2, and rhIL2.

[0073] Figure 19 Shows a series of FACS plots indicating that pan-T cells or Vβ6-5 sorted T cells have high levels of CD25 (CD25 Hi ) or low levels of CD25 (CD25 Lo ). Pan-T cells and Vβ6-5 sorted T cells were expanded using anti-CD3 / CD28 beads and supplemented with recombinant human IL-2. Aliquots of the expanded Vβ6-5+ T cells were stained with PE anti-Vβ6-5 to confirm purity. Aliquots of the expanded pan-T cells and Vβ6-5 T cells with CD25 hi expression were rested for 3 days to allow downregulation of CD25 with CD25 lo expression phenotype.

[0074] Figure 20 Shows that in sorted T cell populations (pan-T cells or Vβ6-5 sorted T cells) that are stimulated and unstimulated, which contain high or low levels of CD25 respectively after anti-CD3 / CD28 stimulation or cell resting, compound 1 binds to Vβ6CD25 Hi and CD25 Lo T cells with higher affinity in a Vβ TCR-dependent manner.

[0075] Figure 21 Shows a gene expression analysis chart of the TRBV-specific T cell lines P12-Ichikawa and HSB-2 via Nanostring, and a gene expression analysis chart of CD25 expression via FACS.

[0076] Figure 22Shows the dose-dependent binding of Compound 1 to the P12-Ichikawa and HSB-2 T cell lines.

[0077] Figure 23 Pictogram showing the relative frequencies of human T cell Vβ6 (purple) and Vβ10 (blue) transcripts before and after stimulation with Compound 1 (n = 3).

[0078] Figure 24 Shows in vitro TCR sequencing. PBMCs were incubated with Compound 1 for 5 days and TCRβ chain V (TRBV) gene sequencing of T cells was performed. Compound 1 selectively amplified T cells bearing TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-5 and TRBV10-3 compared to unstimulated T cells (grey). (n = 3 independent donors).

[0079] Figure 25 Shows a series of FACS plots showing the expansion of Vβ6 / Vβ10 T cells over 8 days.

[0080] Figure 26 Shows a series of plots showing the activation of CD4+ and CD8+ T cells as assessed by CD25 expression after stimulation with Compound 1, or anti-RSV Fab x IL2 control, or anti-Vβ6 / Vβ10 control in solution.

[0081] Figure 27 Shows direct cell counts of CD4+ and CD8+ T cells after stimulation with Compound 1. Mean ± SEM, n = 4.

[0082] Figure 28 Shows purified T cells incubated with Compound 1 and competitive concentrations of soluble IL2R, Vβ6-5 antigen or a mixture of both. Addition of the competitor induced dose-dependent inhibition of T cell activation.

[0083] Figure 29 Shows a series of FACS plots showing the differentiation of Vβ6 / Vβ10 CD8+ T cells mediated by Compound 1 (10 nM) compared to isotype and controls (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 T cells. Asterisks denote plate binding. Cells were treated with Compound 1 or with controls for 7 days prior to analysis.

[0084] Figure 30 Shows a series of plots depicting Figure 23Summary analysis of CD4+ (left) and CD8+ (right) Vβ6 / Vβ10 central memory T cells. Squares represent PBMC, n = 3, and circles represent purified T cells, n = 2.

[0085] Figures 31A-31C Shown is the assessment of TCR and IL-2R signaling using phosphorylation-SLP76, phospho-ERK, and phospho-STAT5 quantification. Figure 31A Shown is that compound 1 increased the pSLP76 level in purified CD8+ T cells compared to those from control molecules. Figure 31B Shown is that compound 1 increased TCR signaling (as measured by the pERK level) compared to the single-arm control. Figure 31C Shown is that compound 1 and the IL-2 control increased IL-2 signaling (as measured by the STAT5 level) compared to the anti-TCRVβ6 / Vβ10 monovalent antibody.

[0086] Figure 32 Is a bar graph showing the percentage of CD25, IFNγ, and granzyme B triple-positive CD8+ T cells after untreated, treated with compound 1 or control.

[0087] Figure 33 Shown is the activation of murine splenocytes cultured with dose-titrated mSTAR, RSV-IL2, and isotype control. Top panel: CD8+ T cells. Bottom panel: CD4+ T cells. Data show one of n = 3 independent donors.

[0088] Figure 34 Shown is the pharmacokinetic profile of mSTAR after a single IP dose of 0.5, 1.0, or 1.5 mg / kg in mice.

[0089] Figure 35 Shown is the pharmacodynamic profile of Vβ13-2 / 3 CD8+ and CD4+ cell subsets and total Tregs after a single IP dose of 1 mg / kg in mice.

[0090] Figure 36 Shown is the biodistribution of mSTAR in tumor, spleen, liver, kidney, and lung tissues of BALB / c EMT6 tumor-bearing mice. Biodistribution was measured at 6, 24, 48, 72, and 120 hours after administration of 1 mg / kg mSTAR. Data are shown as mean + / - SEM.

[0091] Figure 37 Shown is the expansion of Vβ13 T cells after 3 IP doses of 0.5 - 1.5 mg / kg mSTAR in mice, but not after vehicle or rhIL-2.

[0092] Figures 38A-38C Shows the levels of perivascular leukocyte infiltration in mice administered with rhIL2, PBS, or different concentrations of mSTAR. Figure 38A Shows IHC staining of lung and liver tissues, which shows perivascular leukocyte infiltration. Figure 38B Shows the quantification of perivascular CD8+ T cells from liver tissue. Figure 38C Shows the quantification of perivascular CD8+ T cells from lung tissue.

[0093] Figures 39A-39D Shows the changes in serum liver enzyme markers in mice administered with rhIL2, PBS, or mSTAR. Figure 39A Shows aspartate aminotransferase, Figure 39B Shows alanine aminotransferase, Figure 39C Shows alkaline phosphatase, Figure 39D Shows albumin.

[0094] Figures 40A-40B Shows the effects of different doses of mSTAR on tumor volume measurements and mouse survival. mSTAR at increasing concentrations was administered IP once a week for four treatments. Triangles indicate the dosing intervals. n = 8 per group, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns = not significant. Figure 40A Shows that mSTAR at 0.3 mg / kg, 0.5 mg / kg, and 1.0 mg / kg results in potent tumor regression in the EMT6 model. Figure 40B Shows that the potent single-agent activity of mSTAR at 1.0 mg / kg in the EMT6 model has a durable response.

[0095] Figures 41A-41B Shows the effects of a single dose of mSTAR at 1.0 mg / kg on tumor volume measurements and mouse survival. Triangles indicate the dosing times. n = 8 per group, **p < 0.01, *p < 0.05, ns = not significant. Figure 41A Shows that mSTAR results in potent tumor regression in the EMT6 model. Figure 41B Shows that the potent activity of a single dose of mSTAR in the EMT6 model has a durable response.

[0096] Figure 42 Shows the tumor growth curves of mice treated with mSTAR. The study was conducted in random mice with a tumor volume of 80 - 150 mm 3 For all models except RM1, mice were dosed at 1 mg / kg weekly (QW) for 3 - 4 weeks and based on 2000 mm 3Tumor volume endpoint was used to determine survival. For RM1, mice were given a dose of 1.5 mg / kg twice a week (2QW).

[0097] Figure 43 The Kaplan-Meier survival curves of the treated mice are shown. The study was conducted in random mice with a tumor volume of 80 - 150 mm 3 For all models except RM1, mice were dosed at 1 mg / kg once a week (QW) for 3 - 4 weeks, and survival was determined based on a 2000 mm 3 tumor volume endpoint. For RM1, mice were given a dose of 1.5 mg / kg twice a week (2QW).

[0098] Figure 44 The Kaplan-Meier survival curves of the treated mice are shown. The study was conducted in random mice with a tumor volume of 80 - 150 mm 3 For the MC38 and Renca tumor models, mSTAR-treated mice were dosed at 1 mg / kg once a week for 3 weeks. For RM1, mice were given 1.5 mg / kg once a week. For anti-PD1, mice were administered 10 mg / kg anti-mouse PD1 twice a week for a total of five treatments. Survival was determined based on a 2000 mm 3 tumor volume endpoint.

[0099] Figure 45 It is shown that mSTAR induces potent tumor regression in the EMT6 model compared to the single-arm control (n = 8, mean + / - SEM, p < 0.0001).

[0100] Figure 46 IHC staining of EMT6 tumors for CD8 and granzyme B expression is shown.

[0101] Figure 47 Immunophenotyping of CD8+ TIL isolated from EMT6 mice after tumor transplantation is shown. Mean ± SEM, n = 4. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns = not significant.

[0102] Figure 48 Immunophenotyping of NK cells and B cells isolated from EMT6 mice after tumor transplantation is shown. Mean ± SEM, n = 4. ns = not significant.

[0103] Figure 49Shows the immunophenotyping of non-CD8+ TILs isolated from EMT6 mice after tumor transplantation. Mean ± SEM, n = 4. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant.

[0104] Figure 50 Shows the immunophenotyping of CD8+ TILs isolated from EMT6 mice after tumor transplantation comparing RSV F(ab)2x(IL-2)2 and mSTAR administration. Mean ± SEM, n = 4. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant.

[0105] Figure 51 Shows that 1.0 mg / kg mSTAR induces potent tumor regression in the EMT6 model. Depletion of Vβ13 T cells abolishes the anti-tumor activity of mSTAR.

[0106] Figure 52 Shows the results of the tumor rechallenge study. Left: EMT6 tumors are rejected while CT26 tumors grow, indicating that a memory response against EMT6 tumors mediated by mSTAR treatment may have been established. Right: Depletion of CD8+ T cells before rechallenge leads to growth of EMT6 tumors.

[0107] Figure 53 Shows a UMAP plot that depicts the results of single-cell analysis of EMT6 TIL transcriptomes for CD4+ and CD8+ gene expression.

[0108] Figure 54 Shows single-cell RNAseq analysis of single CD4+ or CD8+ TILs isolated from EMT6 mice at day 14 after tumor implantation, after a single dose of mSTAR (right) or vehicle (left), pooled from n = 5 mice per group.

[0109] Figure 55 Shows that Vβ13 T cells are labeled positive based on the gene expression of TRBV13-2 and TRBV13-3.

[0110] Figure 56 Shows the Vβ13 T cells among the UMAP plots inferred from the expression of Figure 54 TRBV13-2 and TRBV13-3 transcripts.

[0111] Figure 57 Shows the 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.

[0112] Figure 58 Shows the quantification of Vβ13 T cells and TIL subtypes.

[0113] Figure 59 Shows a heatmap depicting the number of differentially expressed genes (DEGs) in TILs when comparing the Vβ13+ subset from mSTAR-treated versus vehicle-treated mice.

[0114] Figures 60A-60D Shows a series of volcano plots of differentially expressed genes between the mSTAR-treated targeted Vβ13 T cell subset and the vehicle control treatment group.

[0115] Figure 61 Shows a heatmap of differentially expressed genes in the indicated T cell subsets in response to mSTAR treatment compared to vehicle. The expression values of each gene are scaled.

[0116] Figure 62 Shows that mSTAR induces a novel CD8-T EM phenotype. For each plot, the left violin represents vehicle and the right violin represents mSTAR.

[0117] Figure 63 Shows a heatmap of a series of differentially expressed genes that were identified as unique with mSTAR treatment compared to treatments with IL-2, anti-PD-1, and anti-PD-1-IL-2 mutant proteins from published studies.

[0118] Figure 64 Shows a series of Venn diagrams that display the number of overlapping genes between different compound 1 genes and genes that are significantly and differentially expressed in vehicle and IL-2, anti-PD-1, and anti-PD-1-IL-2 mutant protein treatments.

[0119] Figure 65 Shows a series of violin plots that display TCR signaling repressor genes in CD8 effector T cell subsets and CD8 "better effector" T cell treatments from published studies.

[0120] Figure 66 Shows the clonal diversity of each TRBV gene in TILs obtained from EMT6 mice treated with vehicle (top) or mSTAR (bottom).

[0121] Figure 67It is shown that mSTAR induced an increase in TCR diversity in TIL. Top: Treatment with mSTAR increased clonal diversity in Vβ13-targeted T cells, but not in non-targeted Vβ5 T cells. Bottom: Bubble plots show a substantial increase in unique CDR3 transcripts in Vβ13 TIL treated with mSTAR compared to those treated with vehicle.

[0122] Figure 68 Quantification of TIL clonal diversity between vehicle-treated and mSTAR-treated mice using the inverse Simpson index is shown.

[0123] Figure 69 Single-cell RNAseq analysis of Vβ13 and Vβ5 T cells in vehicle-treated and mSTAR-treated mice is shown.

[0124] Figure 70 Intracellular FACS staining for IFN-γ from an ex vivo tumor antigen recall assay is shown in Vβ13 CD8+ T cell splenocytes isolated from EMT6 tumor-bearing mice treated with 0, 0.5, 1, or 1.5 mg / kg mSTAR.

[0125] Figure 71 Clonal diversity in TIL among MC38 mice treated with vehicle, mSTAR, or anti-RSV-IL-2 is shown. Left: Clone size. Right: Inverse Simpson index of diversity.

[0126] Figure 72 Shows that of CT26 tumor-bearing mice Figure 71 Similar data.

[0127] Figure 73 Tumor-infiltrating lymphocytes (TIL) isolated from CT26 tumor-bearing mice treated with tetramer-stained mSTAR to identify tumor rejection antigen AH1 / gp70 within Vβ13+ CD8+ and Vβ13- CD8+ T cells are shown.

[0128] Figure 74 The pharmacokinetic profile (serum concentration over time) of a single IV dose of Compound 1 in cynomolgus monkeys is shown.

[0129] Figure 75 T cell frequencies in blood after a single IV dose of 1 mg / kg Compound 1 are shown. n = 3 monkeys.

[0130] Figure 76 Serum soluble CD25 levels in monkeys administered a single IV dose of 0.5 mg / kg Compound 1 are shown. Mean ± SEM, n = 6.

[0131] Figure 77 Shows the levels of serum IFNγ, TNFα, and IL-6 in monkeys administered a single IV dose of 0.5 mg / kg of Compound 1. Mean ± SEM, n = 6.

[0132] Figure 78 Shows the levels of serum IL-5 and eosinophil counts in monkeys administered a single IV dose of 1 mg / kg of Compound 1. Mean ± SEM, n = 3.

[0133] Figures 79A-79D Shows the 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 aminotransferase, Figure 79C Shows alkaline phosphatase, Figure 79D Shows albumin.

[0134] Figure 80 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 of Compound 1, 10 μg / ml of pembrolizumab, or 3 μg / ml of isotype control for 5 days.

[0135] Figure 81 Shows the frequency of Vβ6 / Vβ10 T cells among four organoid models.

[0136] Figure 82 Shows the killing of human tumor organoids generated from primary patient-derived tissues from colorectal cancer and NSCLC cancer patients mediated by Compound 1. Bars represent the percentage reduction in organoid area relative to isotype control after incubation of organoids with Compound 1 and autologous TILs. Mean ± SEM, n = 4, ****p < 0.0001, **p < 0.01, ns = not significant.

[0137] Figure 83 Shows the dose-dependent cancer organoid killing by Compound 1 in the NSCLC PDX model. No reduction in organoid size was observed for the IL-2 control molecule.

[0138] Figure 84 Shows the ex vivo activation of HPV-16-specific T cells mediated by Compound 1 in healthy donor PBMCs. Mean ± SEM, n = 7, *p < 0.05, ns = not significant. PBMCs were treated with 1 nM of Compound 1, isotype control, or medium for 1 hour and then stimulated and stained for intracellular expression of IFNγ, TNFα, IL-2, and CD107a with HPV-16 peptide or negative control.

[0139] Figure 85Shows the ex vivo expansion of compound 1-mediated tumor antigen-specific T cells targeting HPV-16. PBMCs were obtained from healthy donors and treated with 1 nM compound 1, anti-TCR Vβ6 / Vβ10 antibody, isotype control, or medium for 1 hour and then stimulated with HPV-16 overlapping 15-mer peptides or negative control for 7 days.

[0140] Figure 86 Shows the 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 peptides or negative control and stained for intracellular expression of IFNγ, TNFα, IL-2, and CD107a.

[0141] Figures 87A-87B Shows compound 1 ( Figure 87A ) and mSTAR ( Figure 87B ) in two SDS-PAGE gels and chromatograms of size exclusion chromatography analysis. Detailed Description

[0142] Definitions

[0143] Certain specific details of this specification are set forth in order to provide a thorough understanding of the 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.

[0144] Unless the context otherwise requires, throughout the specification and the appended claims, the word "comprise" and its variants, such as "comprises" and "comprising", shall be construed in an open, inclusive sense, i.e., "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.

[0145] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. When used in conjunction with the term "comprise", the use of the word "a" or "an" in this context may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0146] It should also be noted that the term "or" is generally used in its inclusive sense of "and / or" unless the context clearly dictates otherwise.

[0147] 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 the present disclosure, suitable methods and materials are described below.

[0148] When referring to measurable values such as amounts, durations, etc., the term "about" is intended to cover variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, such variations being suitable for carrying out the methods of the present disclosure. As used herein, "about" and "approximately" generally refer to an acceptable degree of error of the measured quantity in view of the nature or precision of the given measurement. Exemplary degrees of error are within 20% of the given value range, typically within 10%, and more typically within 5%.

[0149] As used herein, the term "acquire" or "acquiring" means obtaining possession of a physical entity (e.g., a sample, polypeptide, nucleic acid, or sequence) or a value (e.g., a numerical value) by "direct acquisition" or "indirect acquisition". "Direct acquisition" means performing a process (e.g., performing a synthesis or analysis method) to obtain the physical entity or value. "Indirect acquisition" means receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquires the physical entity or value). Direct acquisition of a physical entity includes performing a process that includes a physical change of a physical substance such as a starting material. Direct acquisition of a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process that includes a physical change of a substance (e.g., a sample).

[0150] As used herein, "antibody molecule" refers to a protein that includes at least one immunoglobulin variable domain structure and / or sequence, e.g., an immunoglobulin chain or a fragment thereof. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule includes an antigen-binding fragment 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 a recombinant process from normal immunoglobulin gene segments. In an embodiment, an antibody molecule refers to the immunologically active antigen-binding portion of an immunoglobulin molecule, such as 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 as that recognized by the intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes isolated fragments consisting of the variable region (such as an "Fv" fragment consisting of the variable regions of the heavy and light chains), or recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are joined by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include antibody portions that lack antigen-binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, e.g., dAb (domain antibody), single-chain, Fab, Fab′, and F(ab′)2 fragments, and single-chain variable fragments (scFv). In some embodiments, an antibody molecule is an antibody mimetic. In some embodiments, an antibody molecule is or includes an antibody-like framework or scaffold, such as fibronectin, ankyrin repeats (e.g., designed ankyrin repeat protein (DARPin)), avimer, affibody affinity ligand, anticalin, or affilin molecule.

[0151] As used herein, the term "human-like antibody molecule" refers to a humanized antibody molecule, a human antibody molecule, or an antibody molecule having at least 95% sequence identity with a non-murine germline framework region, e.g., FR1, FR2, FR3, and / or FR4. In some embodiments, a human-like antibody molecule includes 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 an antibody molecule displayed on a phage or yeast. 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.

[0152] As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence can include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence can include or can exclude one, two, or more N- or C-terminal amino acids, or can include other modifications compatible with the formation of the protein structure.

[0153] In embodiments, the antibody molecule is monospecific, e.g., it comprises binding specificity for a single epitope. In some embodiments, the antibody molecule is multifunctional, e.g., it comprises multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence has binding specificity for a first epitope and the second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, a "bispecific antibody molecule" refers to an antibody molecule that is specific for more than one (e.g., two, three, four, or more) epitopes and / or antigens.

[0154] As used herein, an "antigen" (Ag) refers to a molecule that can elicit an immune response (e.g., involving the activation of certain immune cells and / or antibody production). Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein capable of eliciting an immune response encodes an "antigen". In embodiments, the antigen need not be encoded only by the full-length nucleotide sequence of a gene, and the antigen need not be encoded by a gene. In embodiments, the antigen can be synthetic or can be derived from a biological sample, such as a tissue sample, a tumor sample, a cell, or a fluid having other biological components. As used herein, a "tumor antigen" or, interchangeably, a "cancer antigen" includes any molecule present on or associated with a cancer (e.g., cancer cells or tumor microenvironment that can elicit an immune response). As used herein, an "immune cell antigen" includes any molecule present on or associated with an immune cell that can elicit an immune response.

[0155] The "antigen-binding site" or "binding portion" of an antibody molecule refers to the portion of the antibody molecule, such as an immunoglobulin (Ig) molecule, that is involved 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 sequence segments within the variable regions of the heavy and light chains are called hypervariable regions and are located between more conserved flanking sequence segments called "framework regions" (FRs). The FRs are the amino acid sequences that naturally occur between and adjacent to the 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 the bound antigen. The three hypervariable regions in each heavy and light chain are called "complementary determining regions" or "CDRs". The framework regions and CDRs have been defined and described, for example, in Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department 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, which are arranged in the following amino acid sequence from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0156] As used herein, an "immune cell" refers to any one of a variety of cells that function in the immune system to, for example, protect against infectious agents and 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 recognize and eliminate pathogens by attacking larger pathogens via contact or by phagocytosing and killing microorganisms and are mediators of the activation of the adaptive immune response. The cells of the adaptive immune system are a special type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes that are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, while T cells are involved in the cell-mediated immune response. The term "immune cell" includes immune effector cells.

[0157] As used herein, the term "immune effector cell" refers to a cell that participates in an immune response, e.g., a cell that promotes an immune effector response. Examples of immune effector cells include, but are not limited to, T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0158] The term "effector function" or "effector response" refers to the 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.

[0159] The terms "polypeptide", "peptide", and "protein" (if single-stranded) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, such as by 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, can be produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic methods.

[0160] 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, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense) strand. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. Nucleic acids can be recombinant polynucleotides, or polynucleotides from genomic, cDNA, semi-synthetic, or synthetic sources that are not naturally occurring or are linked to another polynucleotide in a non-natural manner.

[0161] As used herein, the term "isolated" refers to a material that has been removed from its source or natural environment (e.g., the natural environment, if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, whereas 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 can be part of a vector, and / or such a polynucleotide or polypeptide can be part of a composition, and still be isolated, because such a vector or composition is not part of the environment in which it is found in nature. Isolated polynucleotides (ribonucleic acid (RNA), deoxyribonucleic acid (DNA)) or polypeptides do not contain the genes / nucleic acids or sequences / amino acids that flank them in their natural state.

[0162] The compositions and methods of the present invention encompass polypeptides and nucleic acids having a specified sequence or a sequence that is substantially the same as or similar to it (e.g., a sequence that is at least 80%, 85%, 90%, 95% identical or higher to the specified sequence). In the context of an amino acid sequence, the term "substantially the same" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues that i) are identical to the aligned amino acid residues in a second amino acid sequence, or ii) are conservative substitutions of the aligned amino acid residues in the second amino acid sequence, such that the first amino acid sequence and the second amino acid sequence can have a common structural domain and / or a common functional activity. For example, an amino acid sequence that contains a common structural domain having 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 (e.g., a sequence provided herein). In the context of a nucleotide sequence, the term "substantially the same" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimal number of nucleotides that are identical to the aligned nucleotides in a second nucleic acid sequence, such that the first nucleotide sequence and the second nucleotide sequence encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, a nucleotide sequence having 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 (e.g., a sequence provided herein).

[0163] The term "variant" refers to a polypeptide having an amino acid sequence that is substantially the same as a reference amino acid sequence or encoded by a substantially the same nucleotide sequence. In some embodiments, the variant is a functional variant. In some embodiments, the TCRβV variant can bind to TCRα and form a TCRα:β complex.

[0164] The term "functional variant" refers to a polypeptide having an amino acid sequence that is substantially the same as a reference amino acid sequence or encoded by a substantially the same nucleotide sequence and capable of having one or more activities of the reference amino acid sequence.

[0165] Homology or sequence identity (these terms are used interchangeably herein) between sequences is calculated as follows. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be disregarded 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%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as at 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").

[0166] The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Mathematical algorithms can be used to accomplish the comparison of sequences and the determination of the percentage identity between two sequences. In a preferred embodiment, the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com)) is used with a Blossum 62 matrix or 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 to determine the percentage identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available from http: / / www.gcg.com) is used with 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 to determine the percentage identity between two nucleotide sequences. A particularly preferred set of parameters (the set of parameters to be used unless otherwise stated) 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.

[0167] The algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), incorporated into the ALIGN program (version 2.0), can be used with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid or nucleotide sequences. The nucleic acid and protein sequences described herein can be used as "query sequences" to search public databases to, for example, identify other family members or related sequences. The searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. A BLAST protein search can be performed using the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402 can be utilized. When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0168] It should be understood that the molecules of the invention can have additional conservative or non-essential amino acid substitutions that do not substantially affect their function.

[0169] The term "amino acid" is intended to cover all molecules, whether natural or synthetic, that contain both an amino functional group and an acid functional group and are capable of being incorporated into a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives, and homologs; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L-enantiomers and peptidomimetics.

[0170] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0171] As used herein, the term "molecule" as used in, for example, antibody molecules, cytokine molecules, receptor molecules includes full-length, naturally occurring molecules, as well as variants, e.g., functional variants (e.g., truncated, fragmented, mutated (e.g., substantially similar sequences) or derivative forms thereof), provided that at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule is retained.

[0172] As used herein, the term "mutation" refers to an alteration in the genomic nucleotide sequence of an organism, virus, or extrachromosomal DNA. In some embodiments, a mutation can be a large-scale mutation, such as amplification (or gene duplication) or duplication of a chromosomal segment, deletion of a large chromosomal region, chromosomal rearrangement (e.g., chromosomal translocation, chromosomal inversion, non-homologous chromosomal crossover, and interstitial deletion), and loss of heterozygosity. In some embodiments, a mutation can be a small-scale mutation, such as insertion, deletion, and substitution mutations. As used herein, the term "substitution mutation" refers to a transition in which a single nucleotide is replaced with another nucleotide.

[0173] "Interleukin-2" (also known as IL2, IL-2, IL 2, TCGF, lymphokine, and interleukin-2) as referred to herein includes any of recombinant or naturally occurring forms of IL-2 or its variants or homologs that have or retain 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, IL-2 is substantially identical to the protein identified by UniProt accession number P60568 or a variant or homolog having substantial identity thereto.

[0174] Anti-TCRβV antibody

[0175] Human T cell receptor (TCR) complex

[0176] The TCR is a disulfide-linked membrane-anchored heterodimeric protein, typically composed of highly variable alpha (α) and beta (β) chains, which are expressed as part of a complex with invariant CD3 chain molecules. The TCR on αβ T cells is formed by a heterodimer of one α chain and one β chain. Each α or β chain consists of a constant domain and a highly variable domain classified as an immunoglobulin superfamily (IgSF) fold. The TCRβV chain can be further divided into 30 subfamilies (TRBV1-30). Although they have high structural and functional homology, the amino acid sequence homology in the TRBV genes is very low. Among approximately 95 amino acids, only 4 amino acids are identical, and 10 additional amino acids are conserved among all subfamilies. However, the TCR formed between highly diverse α and β chains shows significant structural homology and elicits similar functions, e.g., activation of T cells.

[0177] T cell receptors (TCRs) can be found on the surface of T cells. TCRs recognize antigens (e.g., peptides) presented (e.g., bound) on major histocompatibility complex (MHC) molecules on the surface of cells (e.g., antigen-presenting cells). TCRs are heterodimeric molecules and can consist of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain, or a delta (δ) chain. TCRs containing an α chain and a β chain are also referred to as TCRαβ. The TCR β chain consists of the following regions (also called segments): variable (V), diverse (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 on-line, University of South Carolina School of Medicine). The TCR α chain consists of a V region, a J region, and a C region. T cell receptor (TCR) rearrangement by somatic recombination of variable (V), diverse (D), joining (J), and constant (C) regions is a decisive event in T cell development and maturation. TCR gene rearrangement occurs in the thymus.

[0178] TCRs can contain a receptor complex called the TCR complex, which includes a TCR heterodimer composed of an α chain and a β chain and dimeric signaling molecules, such as CD3 coreceptors, such as CD3δ / ε and / or CD3γ / ε.

[0179] 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 includes the antigen recognition domain of the T cell receptor. The term TCRβV includes isoforms, mammalian (e.g., human) TCRβV, human homologs, and analogs that have at least one common epitope with TCRβV. Human TCRβV includes gene families that include subfamilies including, but not limited to: TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily, TCRβV29 subfamily, TCRβV1 subfamily, TCRβV17 subfamily, TCRβV21 subfamily, TCRβV23 subfamily or TCRβV26 subfamily, and family members of said subfamilies, and variants thereof (e.g., structural or functional variants). In some embodiments, the TCRβV6 subfamily includes: TCRβV6-4*01, TCRβV6-4*02, TCRβV6-9*01, TCRβV6-8*01, TCRβV6-5*01, TCRβV6-6*02, TCRβV6-6*01, TCRβV6-2*01, TCRβV6-3*01 or TCRβV6-1*01. In some embodiments, TCRβV includes TCRβV6-5*01 or a variant thereof, e.g., a variant having 85%, 90%, 95%, 99% or higher identity to the naturally occurring sequence. TCRβV6-5*01 is also known as TRBV65; TCRBV6S5; TCRBV13S1 or TCRβV13.1. The amino acid sequence of TCRβV6-5*01, e.g., human TCRβV6-5*01, is known in the art, e.g., as provided by IMGT ID L36092. In some embodiments, TCRβV6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO:43 or a sequence having 85%, 90%, 95%, 99% or higher identity thereto. In some embodiments, TCRβV6-5*01 includes the amino acid sequence of SEQ ID NO:44, or a sequence having 85%, 90%, 95%, 99% or higher identity thereto.

[0180] SEQ ID NO:43

[0181] ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTC

[0182] SEQ ID NO:44

[0183] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHY-SVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY

[0184] TCRβV(TCRβV)

[0185] The diversity of the immune system enables protection against a large number of pathogens. Due to the limited size of the germline genome, diversity is achieved not only through the process of V(D)J recombination, but also through nucleotide joining (joining between V-D and D-J segments), deletions, and the addition of pseudo-random non-template nucleotides. The TCRβ gene generates diversity through gene rearrangement.

[0186] The TCR Vβ repertoire varies among individuals and populations due to, for example, the frequent occurrence of 7 inactivating polymorphisms in functional gene segments and large insertion / deletion-related polymorphisms spanning 2 Vβ gene segments.

[0187] The present invention particularly provides antibody molecules and fragments thereof that bind (e.g., specifically bind) to the human TCRβ variable chain (TCRβV), such as TCRβV gene families (also referred to as groups), such as TCRβV subfamilies (also referred to as subgroups), such as as described herein. TCRβV families and subfamilies are known in the art, e.g., as described 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 can be recombinant antibodies, e.g., recombinant non-mouse antibodies, e.g., recombinant human or humanized antibodies.

[0188] The terms TCRBV, TCRVB, TRBV, TCRβV, TCRVβ or TRβV are used interchangeably herein and refer to the TCRβV chain, e.g., as described herein.

[0189] In some embodiments, the present invention provides anti-TCRβV antibody molecules that bind to human TCRβV, such as TCRβV families, such as gene families, or variants thereof. In some embodiments, the TCRBV gene family comprises one or more subfamilies, such as as described herein, e.g., in Table 8A or Table 8B. In some embodiments, the TCRβV gene family comprises: the TCRβV6 subfamily or the TCRβV10 subfamily.

[0190] In some embodiments, the TCRβV6 subfamily is also referred to as TCRβV13.1. In some embodiments, the TCRβV6 subfamily includes: TCRβV6-4*01, TCRβV6-4*02, TCRβV6-9*01, TCRβV6-8*01, TCRβV6-5*01, TCRβV6-6*02, TCRβV6-6*01, TCRβV6-2*01, TCRβV6-3*01, or TCRβV6-1*01, or variants thereof. In some embodiments, TCRβV6 includes TCRβV6-4*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-4*02 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-9*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-8*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-5*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-6*02 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-6*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-2*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-3*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-1*01 or a variant thereof.

[0191] In some embodiments, TCRβV6 includes TCRβV6-5*01 or a variant thereof. In some embodiments, TCRβV6, such as TCRβV6-5*01, is recognized, such as bound, by SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, TCRβV6, such as TCRβV6-5*01, is recognized, such as bound, by SEQ ID NO:9 and / or SEQ ID NO:10. In some embodiments, TCRβV6 is recognized, such as bound, by SEQ ID NO:9 and / or SEQ ID NO:11.

[0192] In some embodiments, the TCRβV10 subfamily includes: TCRβV10-1*01, TCRβV10-1*02, TCRβV10-3*01, or TCRβV10-2*01, or variants thereof.

[0193] Exemplary amino acid sequences of TCRβV subfamily members can be found at the ImMunoGeneTics Information System website: http: / / www.imgt.org / or similar resources.

[0194] Anti-TCRβV antibody

[0195] Current bispecific constructs designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically utilize antibody fragments (Fab, scFv, VH, single-domain antibodies, etc.) derived from monoclonal antibodies (mAbs) against the CD3e subunit of the T cell receptor (TCR). However, limitations of this approach may limit 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 caused by large-scale T cell activation. A large number of activated T cells secrete large amounts of cytokines, the most important of which is interferon gamma (IFNγ). This excess IFNγ then activates macrophages, which then overproduce pro-inflammatory cytokines such as IL-1β, IL-6, IL-10, and TNF-α, causing a "cytokine storm" known as cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., J Immunother Cancer. June 15, 2018; 6(1):56, which is incorporated herein by reference in its entirety). Therefore, there is a need to develop antibodies that can bind and activate only effector T cell subsets, for example, to reduce CRS and / or neurotoxicity (NT).

[0196] Described herein are molecules and methods that target the TCRβV chain of the TCR. Without wishing to be bound by theory, such molecules are capable of binding, activating, and / or expanding only subsets of T cells, avoiding or reducing CRS and / or NT, and minimizing the potential immunosuppressive effects of anti-CD3 mAbs.

[0197] Described herein is a class of antibodies, namely anti-TCRβV antibody molecules as described herein, that recognize structurally conserved but sequence-variable regions, such as domains, on the TCRβV protein and have similar functions (e.g., T cell activation and similar cytokine profiles as described herein), despite having low sequence similarity (e.g., low sequence identity between different antibody molecules that recognize different TCRβV subfamilies). Thus, anti-TCRβV antibody molecules as described herein share structure-function relationships.

[0198] Without wishing to be bound by theory, in some embodiments, when an anti-TCRβV antibody molecule as described herein is in complex with a TCRα protein, it binds to an outward-facing epitope of the TCRβV protein. In some embodiments, an anti-TCRβV antibody molecule as described herein recognizes (e.g., binds to) a domain (e.g., an epitope) on the TCRβV protein that: (1) is structurally conserved among different TCRβV subfamilies; and (2) has minimal sequence identity among different TCRβV subfamilies. TCRβV proteins from different TCRBV subfamilies share minimal sequence similarity. However, TCRβV proteins with minimal sequence similarity share similar 3D conformations and structures.

[0199] Alignment of TCRBV amino acid sequences highlights the diversity of TCR sequences. Specifically, TRBV sequences from different subfamilies are quite different from each other.

[0200] In some embodiments, an anti-TCRβV antibody molecule as described herein does not recognize, e.g., does not bind to the interface of the TCRβV:TCRα complex. In some embodiments, an anti-TCRβV antibody molecule as described herein does not recognize, e.g., does not bind to the constant region of the TCRβV protein. An exemplary antibody that binds to the constant region of the TCRBV region is JOVI.1, as described by Viney et al. (Hybridoma. December 1992; 11(6):701-13). In some embodiments, an anti-TCRβV antibody molecule as described herein does not recognize, e.g., does not bind to one or more (e.g., all) complementarity-determining regions (e.g., CDR1, CDR2, and / or CDR3) of the TCRβV protein.

[0201] The present invention particularly provides antibody molecules directed against the variable chain of the β subunit (TCRβV) of the TCR, which bind to and, for example, activate subsets of T cells. An anti-TCRβV antibody molecule as described herein results in less or no production of cytokines associated with CRS, such as IL-6, IL-1β, IL-10, and TNFα; and enhanced and / or delayed production of IL-2 and IFNγ. In some embodiments, an anti-TCRβV antibody as described herein has, for example, a cytokine profile as described herein that is different from the cytokine profile of a T cell engager that binds to a receptor or molecule other than the TCRβV region ("non-TCRβV-binding T cell engager"). In some embodiments, a non-TCRβV-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., a CD3ε (CD3e) molecule), or a TCRα (TCRα) molecule. In some embodiments, a non-TCRβV-binding T cell engager is an OKT3 antibody or an SP34-2 antibody.

[0202] In some embodiments, the anti-TCRβV antibodies as described herein result in the expansion of TCRβV+ T cells (e.g., a subset of memory effector T cells called TEMRA). Without being bound by theory, it is believed that in some embodiments, TEMRA cells can promote tumor cell lysis but not CRS. Accordingly, methods of making the anti-TCRβV antibody molecules and their uses are provided herein. Multifunctional molecules, such as bispecific molecules comprising the anti-TCRβV antibody molecules, are also described herein. In some embodiments, a composition comprising an anti-TCRβV antibody molecule of the present disclosure can be used for, e.g.: (1) activating and redirecting T cells to promote tumor cell lysis for cancer immunotherapy; and / or (2) expanding TCRβV+ T cells. In some embodiments, a composition comprising an anti-TCRβV antibody molecule as described herein limits the deleterious side effects of CRS and / or NT (e.g., CRS and / or NT associated with anti-CD3e targeting).

[0203] 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, 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.

[0204] 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.

[0205] In some embodiments, the light or heavy chain variable frameworks (e.g., regions comprising at least FR1, FR2, FR3, and optionally FR4) of anti-TCRβV antibody molecules, such as anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecules, can be selected from: (a) light or heavy chain variable frameworks comprising at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or 100% amino acid residues from human light or heavy chain variable frameworks, such as light or heavy chain variable framework residues from human mature antibodies, germline sequences, or human consensus sequences; (b) light or heavy chain variable frameworks comprising 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% amino acid residues from human light or heavy chain variable frameworks, such as light or heavy chain variable framework residues from human mature antibodies, germline sequences, or human consensus sequences; (c) non-human frameworks (e.g., rodent frameworks); or (d) non-human frameworks that have been modified, such as by removing antigenic or cytotoxic determinants, e.g., deimmunized or partially humanized non-human frameworks. In some embodiments, the light or heavy chain variable framework regions (particularly FR1, FR2, and / or FR3) comprise a light or heavy chain variable framework sequence that is the same as or at least 70%, 75%, 80%, 85%, 87%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to the framework of the VL or VH segment of a human germline gene.

[0206] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a heavy chain variable domain that has at least one, two, three, four, five, six, seven, ten, fifteen, twenty, or more changes, such as amino acid substitutions or deletions, compared to the amino acid sequence of any one of A-H.1 to A-H.85 (e.g., A-H.1, A-H.2, or A-H.68, e.g., the amino acid sequence of the FR regions in the entire variable region, e.g., as shown in SEQ ID NO:9).

[0207] Alternatively, or in combination with the heavy chain replacements described herein, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that has at least one, two, three, four, five, six, seven, ten, fifteen, twenty, or more amino acid changes, such as amino acid substitutions or deletions, compared to the amino acid sequence of any one of A-H.1 to A-H.85 (e.g., A-H.1, A-H.2, or A-H.68, e.g., the amino acid sequence of the FR regions in the entire variable region, e.g., as shown in SEQ ID NO:10 or SEQ ID NO:11).

[0208] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises light chain framework region 1 of A-H.1 or A-H.2.

[0209] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises light chain framework region 2 of A-H.1 or A-H.2.

[0210] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises light chain framework region 3 of A-H.1 or A-H.2.

[0211] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises light chain framework region 4 of A-H.1 or A-H.2.

[0212] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that comprises a framework region, such as framework region 1 (FR1), the framework region comprising a change, such as a substitution (e.g., a conservative substitution) at position 10 according to Kabat numbering. In some embodiments, FR1 comprises phenylalanine at position 10, such as a substitution of serine to phenylalanine. In some embodiments, the substitution is relative to the germline light chain framework region sequence.

[0213] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that comprises a framework region, such as framework region 2 (FR2), the framework region comprising a change, such as a substitution (e.g., a conservative substitution) at a position as described herein according to Kabat numbering. In some embodiments, FR2 comprises histidine at position 36, such as a substitution at position 36 according to Kabat numbering, such as a substitution of tyrosine to histidine. In some embodiments, FR2 comprises alanine at position 46, such as a substitution at position 46 according to Kabat numbering, such as a substitution of arginine to alanine. In some embodiments, the substitution is relative to the germline light chain framework region sequence.

[0214] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that includes a framework region, such as framework region 3 (FR3), the framework region including alterations, such as substitutions (e.g., conservative substitutions) at positions as described herein according to Kabat numbering. In some embodiments, FR3 comprises phenylalanine at position 87, such as a substitution at position 87 according to Kabat numbering, such as a substitution of tyrosine to phenylalanine. In some embodiments, the substitution is relative to the germline light chain framework region sequence.

[0215] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that includes: (a) framework region 1 (FR1) that comprises phenylalanine at position 10, such as a substitution at position 10 according to Kabat numbering, such as a substitution of serine to phenylalanine; (b) framework region 2 (FR2) that comprises histidine at position 36, such as a substitution at position 36 according to Kabat numbering, such as a substitution of tyrosine to histidine, and comprises alanine at position 46, such as a substitution at position 46 according to Kabat numbering, such as a substitution of arginine to alanine; and (c) framework region 3 (FR3) that comprises phenylalanine at position 87, such as a substitution at position 87 according to Kabat numbering, such as a substitution of tyrosine to phenylalanine, such as as shown by the amino acid sequence of SEQ ID NO:10. In some embodiments, the substitution is relative to the germline light chain framework region sequence.

[0216] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that includes: (a) framework region 2 (FR2) that comprises histidine at position 36, such as a substitution at position 36 according to Kabat numbering, such as a substitution of tyrosine to histidine, and comprises alanine at position 46, such as a substitution at position 46 according to Kabat numbering, such as a substitution of arginine to alanine; and (b) framework region 3 (FR3) that comprises phenylalanine at position 87, such as a substitution at position 87 according to Kabat numbering, such as a substitution of tyrosine to phenylalanine, such as as shown by the amino acid sequence of SEQ ID NO:11. In some embodiments, the substitution is relative to the germline light chain framework region sequence.

[0217] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a light chain variable domain that includes: (a) framework region 1 (FR1) that includes alterations, such as substitutions (e.g., conservative substitutions), at one or more (e.g., all) positions as described herein according to Kabat numbering; (b) framework region 2 (FR2) that includes alterations, such as substitutions (e.g., conservative substitutions), at one or more (e.g., all) positions as described herein according to Kabat numbering; and (c) framework region 3 (FR3) that includes alterations, such as substitutions (e.g., conservative substitutions), at one or more (e.g., all) positions as described herein according to Kabat numbering. In some embodiments, the substitutions are relative to the germline light chain framework region sequence.

[0218] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes heavy chain framework region 1 of A-H.1 or A-H.2. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes heavy chain framework region 2 of A-H.1 or A-H.2. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes heavy chain framework region 3 of A-H.1 or A-H.2. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes heavy chain framework region 4 of A-H.1 or A-H.2.

[0219] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a heavy chain variable domain that includes a framework region, such as framework region 3 (FR3), that includes an alteration, such as a substitution (e.g., a conservative substitution) at a position as described herein according to Kabat numbering. In some embodiments, FR3 includes threonine at position 73, such as a substitution at position 73 according to Kabat numbering, such as a substitution of glutamate to threonine. In some embodiments, FR3 includes glycine at position 94, such as a substitution at position 94 according to Kabat numbering, such as a substitution of arginine to glycine. In some embodiments, the substitutions are relative to the germline heavy chain framework region sequence.

[0220] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a heavy chain variable domain that comprises framework region 3 (FR3) that comprises threonine at position 73, such as a substitution at position 73 according to Kabat numbering, such as a substitution of glutamate to threonine, and that comprises glycine at position 94, such as a substitution at position 94 according to Kabat numbering, such as a substitution of arginine to glycine, e.g., as shown in the amino acid sequence of SEQ ID NO:10.

[0221] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises heavy chain framework regions 1-4 of A-H.1 or A-H.2, such as SEQ ID NO:9. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises light chain framework regions 1-4 of A-H.1, such as SEQ ID NO:10. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises light chain framework regions 1-4 of A-H.2, such as SEQ ID NO:11. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises heavy chain framework regions 1-4 of A-H.1, such as SEQ ID NO:9; and light chain framework regions 1-4 of A-H.1, such as SEQ ID NO:10. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises heavy chain framework regions 1-4 of A-H.2, such as SEQ ID NO:9; and light chain framework regions 1-4 of A-H.2, such as SEQ ID NO:11.

[0222] In some embodiments, the heavy chain or light chain variable domain or both of an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises an amino acid sequence that is substantially the same as an amino acid described herein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to the variable region of an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2 or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence of Table 1); or differs from the variable region of an antibody described herein by at least 1 or 5 residues, but by fewer than 40, 30, 20 or 10 residues.

[0223] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, three, or four antigen-binding regions (e.g., variable regions) having an amino acid sequence as 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 thereto, or a sequence that differs from the sequence shown in Table 1 by no more than 1, 2, 5, 10, or 15 amino acid residues). In another embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises VH and / or VL domains encoded by a nucleic acid having a nucleotide sequence as 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 thereto, or a sequence that differs from the sequence shown in Table 1 by no more than 3, 6, 15, 30, or 45 nucleotides).

[0224] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:9, an amino acid sequence that is 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 that is 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.

[0225] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises: a VH domain that comprises the amino acid sequence of SEQ ID NO:9, an amino acid sequence that is 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 that comprises the amino acid sequence of SEQ ID NO:11, an amino acid sequence that is 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.

[0226] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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 an embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is a monoclonal antibody or an antibody with a single specificity. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, may also be a humanized, chimeric, camelid, shark or in vitro-produced antibody molecule. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is a humanized antibody molecule. The heavy and light chains of an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, may be full-length (e.g., the antibody may comprise at least one, preferably two full-length heavy chains, and at least one, preferably two full-length light chains) or may comprise antigen-binding fragments (e.g., Fab, F(ab’)2, Fv, single-chain Fv fragment, single-domain antibody, diabody (dAb), bivalent antibody, or bispecific antibody or a fragment thereof, a single-domain variant thereof or a camelid antibody).

[0227] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is in the form of a multifunctional molecule, such as a bispecific molecule, as described herein.

[0228] In some embodiments, the anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, has a heavy chain constant region (Fc) selected from the following: the heavy chain constant regions of, for example, 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 regions 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, such as, as described herein.

[0229] In some embodiments, the anti-TCRβV antibody molecule, e.g., an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, has a light chain constant region selected from the following: the light chain constant regions of, for example, κ or λ, preferably κ (e.g., human κ). In some embodiments, the constant region is altered, such as mutated to modify the properties of the anti-TCRβV antibody molecule (e.g., an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule) (e.g., to increase or decrease one or more of the following: 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., 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, e.g., relative to human IgG1).

[0230] Antibody A-H.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 A-H.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 A-H.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 A-H.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.

[0231] Additional exemplary humanized anti-TCRB V6 antibodies are provided in Table 1. In some embodiments, the anti-TCRβV6 is Antibody A, such as humanized Antibody A (Antibody A-H), as provided in Table 1. In some embodiments, the anti-TCRβV antibody comprises one or more (e.g., all three) of LC CDR1, LC CDR2, and LC CDR3 provided in Table 1; and / or one or more (e.g., all three) of 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 the 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.

[0232] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a VH of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.41, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84 or A-H.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0233] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a VL of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.41, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84 or A-H.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0234] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a VH of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.1, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84 or A-H.85, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto; and A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.1, A-H.42, A-H.43, A-H.44, A-H.45, A-H.VL of 46, A-H; 47, A-H; 48, A-H; 49, A-H; 50, A-H; 51, A-H; 52, A-H; 53, A-H; 54, A-H; 55, A-H; 56, A-H; 57, A-H; 58, A-H; 59, A-H; 60, A-H; 61, A-H; 62, A-H; 63, A-H; 64, A-H; 65, A-H; 66, A-H; 67, A-H; 68, A-H; 69, A-H; 70, A-H; 71, A-H; 72, A-H; 73, A-H; 74, A-H; 75, A-H; 76, A-H; 77, A-H; 78, A-H; 79, A-H; 80, A-H; 81, A-H; 82, A-H; 83, A-H; 84, A-H or 85, A-H, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0235] Exemplary anti-TCRβV antibody molecules and the corresponding TCRβV subfamilies recognized by the anti-TCRβV antibody molecules are disclosed in Table 10A.

[0236] Various TCRβV subfamilies and / or subfamily members can be expressed at different levels in an individual (e.g., a healthy individual), as disclosed by Kitaura K. et al. (2016), BMC Immunology, Vol. 17:38, the entire content of which is incorporated herein by reference. For example, TCRβV6-5 is present in approximately 3-6% of healthy donors.

[0237] The manifestations of various TCRBV subfamilies and / or subfamily members may also be different in cancer cells. For example, TCRβV is present in approximately 3-6% of tumor-infiltrating T cells, regardless of the tumor type (see Li B. et al., Nature Genetics, 2016, Vol. 48(7):725-32, the entire content of which is incorporated herein by reference). Li et al. also disclosed that TCRβV6-5 is present at a high frequency in tumor cells.

[0238] Anti-TCRβV6 antibody

[0239] In one aspect, provided herein are anti-TCRβV antibody molecules that bind to human TCRβV6 (e.g., the TCRβV6 subfamily), which TCRβV6 subfamily includes: TCRβV6-4*01, TCRβV6-4*02, TCRβV6-9*01, TCRβV6-8*01, TCRβV6-5*01, TCRβV6-6*02, TCRβV6-6*01, TCRβV6-2*01, TCRβV6-3*01, or TCRβV6-1*01. In some embodiments, the TCRβV6 subfamily includes TCRβV6-5*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-4*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-4*02 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-9*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-8*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-5*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-6*02 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-6*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-2*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-3*01 or a variant thereof. In some embodiments, TCRβV6 includes TCRβV6-1*01 or a variant thereof.

[0240] In some embodiments, TCRβV6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO:43 or a sequence having 85%, 90%, 95%, 99% or higher identity thereto. In some embodiments, TCRβV6-5*01 comprises the amino acid sequence of SEQ ID NO:44, or an amino acid sequence having 85%, 90%, 95%, 99% or higher identity thereto.

[0241] In some embodiments, the anti-TCRβV antibody molecule, e.g., an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is a non-mouse antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is a human antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is a humanized antibody molecule.

[0242] In some embodiments, the anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, is isolated or recombinant.

[0243] In some embodiments, the anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one antigen-binding region, such as a variable region or an antigen-binding fragment thereof, from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2 or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in 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 above sequences).

[0244] In some embodiments, the anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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 A-H.1 to A-H.85, such as A-H.1, A-H.2 or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in 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 above sequences).

[0245] In some embodiments, the anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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 A-H.1 to A-H.85, such as A-H.1, A-H.2 or A-H.68, or an antibody molecule described in Table 1, or an antibody encoded by the nucleotide sequence in 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 above sequences).

[0246] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one or two light chain variable regions from an antibody described herein (e.g., an antibody selected from any of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in Table 1, or a sequence that is substantially the same as any of the foregoing sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)).

[0247] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a heavy chain constant region of IgG4 (e.g., human IgG4). In yet another embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a heavy chain constant region of IgG1 (e.g., human IgG1). In some embodiments, the heavy chain constant region comprises the amino sequence shown in Table 3, or a sequence that is substantially the same as it (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0248] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a κ light chain constant region, such as a human κ light chain constant region. In some embodiments, the light chain constant region comprises the amino sequence listed in Table 3 or a sequence that is substantially the same as it (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

[0249] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three complementarity-determining regions (CDRs) of the heavy chain variable region (VH) from an antibody described herein (e.g., an antibody selected from any of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in Table 1, or a sequence that is substantially the same as any of the foregoing sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical)).

[0250] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or all CDRs) from a heavy chain variable region comprising the amino acid sequence shown in Table 1 or an amino acid sequence encoded by the nucleotide sequence shown in Table 1. In some embodiments, relative to the amino acid sequence shown in Table 1 or an amino acid sequence encoded by the nucleotide sequence shown in Table 1, one or more CDRs (or all CDRs) have one, two, three, four, five, six, or more alterations, such as amino acid substitutions or deletions.

[0251] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three complementarity-determining regions (CDRs) from a light chain variable region of an antibody described herein (e.g., an antibody selected from any of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in Table 1, or a sequence that is substantially the same (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical) to any of the foregoing sequences).

[0252] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three CDRs (or all CDRs) from a light chain variable region comprising the amino acid sequence shown in Table 1 or an amino acid sequence encoded by the nucleotide sequence shown in Table 1. In some embodiments, relative to the amino acid sequence shown in Table 1 or an amino acid sequence encoded by the nucleotide sequence shown in Table 1, one or more CDRs (or all CDRs) have one, two, three, four, five, six, or more alterations, such as amino acid substitutions or deletions.

[0253] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, three, four, five, or six CDRs (or all CDRs) from a heavy and light chain variable region comprising the amino acid sequence shown in Table 1 or an amino acid sequence encoded by the nucleotide sequence shown in Table 1. In some embodiments, relative to the amino acid sequence shown in Table 1 or an amino acid sequence encoded by the nucleotide sequence shown in Table 1, one or more CDRs (or all CDRs) have one, two, three, four, five, six, or more alterations, such as amino acid substitutions or deletions.

[0254] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, the molecule comprises all six CDRs from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in Table 1), or comprises closely related CDRs, such as CDRs that are identical or have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions). In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule may include any CDR described herein.

[0255] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs defined according to Kabat as listed in Table 1) of the heavy chain variable region from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in 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 above sequences); or the CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1.

[0256] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs defined according to Kabat as listed in Table 1) of the light chain variable region from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in 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 above sequences); or the CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1.

[0257] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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 as defined by Kabat in Table 1) of the heavy and light chain variable regions from an antibody described herein (e.g., an antibody selected from any of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in Table 1; or a sequence that is substantially the same as any of the foregoing sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical)); or the CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conservative substitution) relative to the six CDRs according to Kabat et al. shown in Table 1.

[0258] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises all six CDRs according to Kabat et al. (e.g., all six CDRs as defined by Kabat in Table 1) of the heavy and light chain variable regions from an antibody described herein (e.g., an antibody selected from any of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequence in Table 1; or a sequence that is substantially the same as any of the foregoing sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical)); or the CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitution, deletion, or insertion, such as a conservative substitution) relative to the six CDRs according to Kabat et al. shown in Table 1. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, can include any CDR described herein.

[0259] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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 of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68), e.g., a canonical structure that is the same as at least loop 1 and / or loop 2 of the heavy and / or light chain variable domains of an antibody described herein. For a description of hypervariable loop canonical structures, see, e.g., Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798. These structures can be determined by examining the tables described in these references.

[0260] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three CDRs according to Chothia et al. of the heavy chain variable region from an antibody described herein (e.g., an antibody selected from any of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or as described in Table 1, or a sequence that is substantially the same as any of the foregoing sequences (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more identical)); or said CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in Table 1.

[0261] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs as defined by Chothia listed in Table 1) of the light chain variable region from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.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 the CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in Table 1.

[0262] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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 as defined by Chothia listed in Table 1) of the heavy and light chain variable regions from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequences 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 the CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Chothia et al. shown in Table 1.

[0263] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises all six CDRs according to Chothia et al. (e.g., all six CDRs defined according to Chothia as listed in Table 1) of the heavy and light chain variable regions from an antibody described herein (e.g., an antibody selected from any one of A-H.1 to A-H.85, such as A-H.1, A-H.2, or A-H.68, or an antibody described in Table 1, or an antibody encoded by the nucleotide sequences in 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 above sequences); or said CDRs have at least one amino acid change but no more than two, three, or four changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions) relative to all six CDRs according to Chothia et al. shown in Table 1. In one embodiment, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, may include any CDR described herein.

[0264] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a combination of CDRs or hypervariable loops defined according to Kabat et al., Chothia et al., or as described in Table 1.

[0265] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, may comprise any combination of CDRs or hypervariable loops defined according to Kabat and Chothia.

[0266] In some embodiments, the CDRs of the combination listed in Table 1 are CDRs that include Kabat CDRs and Chothia CDRs.

[0267] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises a combination of CDRs or hypervariable loops that are identified in Table 1 as the combined CDRs. In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, may comprise any combination of CDRs or hypervariable loops of the "combined" CDRs as described in Table 1.

[0268] In some embodiments, such as embodiments that include a variable region, a CDR (e.g., a combined CDR, a Chothia CDR, or a Kabat CDR), or other sequences mentioned herein, such as in Table 1 for example, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a bispecificity antibody molecule, or an antibody molecule that includes 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 includes a multifunctional molecule, such as a bispecific molecule, as described herein for example.

[0269] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, includes: (i) one, two, or all of the 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 the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HCCDR2), and heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO:1 or SEQ ID NO:9.

[0270] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO:2, and the HC CDR1, HCCDR2, and HC CDR3 of SEQ ID NO:1.

[0271] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO:10, and the HC CDR1, HCCDR2, and HC CDR3 of SEQ ID NO:9.

[0272] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO:11, and the HC CDR1, HCCDR2, and HC CDR3 of SEQ ID NO:9.

[0273] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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.

[0274] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises: (i) a light chain variable region (VL) that comprises 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) that comprises 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.

[0275] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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.

[0276] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises: (i) a light chain variable region (VL) that comprises 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) that comprises 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.

[0277] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) 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.

[0278] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises: (i) a light chain variable region (VL) that comprises 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) that comprises 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.

[0279] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises the VH and / or VL of the antibodies described in Table 1, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0280] In some embodiments, an anti-TCRβV antibody molecule, such as an anti-TCRβV6 (e.g., anti-TCRβV6-5*01) antibody molecule, comprises the VH and VL of the antibodies described in Table 1, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0281] In some embodiments, an anti-TCRVb antibody as described herein has an antigen-binding domain that has a VL containing 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.

[0282] In some embodiments, an anti-TCRVb antibody as described herein has an antigen-binding domain having a VH containing the consensus sequence of SEQ ID NO:231, wherein: position 27 is H or T or G or Y; position 28 is D or T or S; position 30 is H or R or D or K or T; position 31 is L or D or K or T or N; position 32 is W or F or T or I or Y or G; position 49 is R or W; position 50 is V or I or F; position 51 is F or S or Y; position 52 is A or P; position 56 is N or S; position 57 is T or V or 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.

[0283] Anti-TCRβV10 antibody

[0284] In one aspect, the present disclosure provides anti-TCRβV antibody molecules that bind to members of the human TCRβV10 subfamily. In some embodiments, the TCRβV10 subfamily is also referred to as TCRβV12. In some embodiments, the TCRβV10 subfamily includes: TCRβV10-1*01, TCRβV10-1*02, TCRβV10-3*01, or TCRβV10-2*01, or variants thereof.

[0285] Exemplary anti-TCRβV10 antibodies are provided in Table 12. In some embodiments, anti-TCRβV10 is antibody D, such as humanized antibody D (antibody D-H), as 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 having at least 95% sequence identity thereto. In some embodiments, antibody D comprises the variable heavy chain (VH) and / or variable light chain (VL) provided in Table 12, or a sequence having at least 95% sequence identity thereto.

[0286] In some embodiments, an anti-TCRβV10 antibody molecule comprises the VH or VL of the antibody described in Table 12, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0287] In some embodiments, an anti-TCRβV10 antibody molecule comprises the VH and VL of the antibody described in Table 12, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0288] Antibody-like frameworks or scaffolds

[0289] A variety of antibody / immunoglobulin frameworks or scaffolds can be used for anti-TCRvb antibody molecules or their multifunctional forms as described herein, provided that the resulting polypeptide includes at least one binding region that specifically binds to a target antigen such as TCRvb, tumor antigen, etc. Such frameworks or scaffolds include the 5 major unique types of human immunoglobulins or fragments thereof, and include immunoglobulins from other animal species preferably having humanized aspects. Those skilled in the art will continue to discover and develop novel frameworks, scaffolds, and fragments.

[0290] In some embodiments, the anti-TCRvb antibody molecules or their multifunctional forms as described herein include non-immunoglobulin-based antibodies that employ non-immunoglobulin scaffolds onto which CDRs can be grafted. Any non-immunoglobulin framework and scaffold can be used as long as they contain a binding region that is 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 antibody (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceutical (Trubion Pharmaceuticals Inc., Seattle, WA), maxybody (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden), and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmB-H, Halle, Germany).

[0291] Fibronectin scaffolds are generally based on fibronectin type III domains (e.g., the tenth module of fibronectin type III (10Fn3 domain)). Fibronectin type III domains have 7 or 8 β-strands distributed between two β-sheets, which stack on themselves to form the core of the protein, and further contain loops (similar to CDRs) that connect the β-strands to each other and are exposed to the solvent. There are at least three such loops on each edge of the β-sheet sandwich structure, where the edge is the boundary of the protein perpendicular to the direction of the β-strands (see US 6,818,418). Due to this structure, non-immunoglobulin antibodies mimic antigen-binding properties similar to antibodies in nature and affinity. These scaffolds can be used for in vitro loop randomization and shuffling strategies similar to the process of in vivo antibody affinity maturation. These fibronectin-based molecules can be used as scaffolds, where the loop regions of the molecules can be replaced with the CDRs of the present invention using standard cloning techniques.

[0292] Ankyrin technology is based on using proteins with ankyrin-derived repeat modules as scaffolds with variable regions that can be used to bind different targets. Ankyrin repeat modules are generally polypeptides of about 33 amino acids consisting of two anti-parallel α-helices and a β-turn. The binding of the variable region can be optimized by using ribosome display.

[0293] Avimers are used for protein-protein interactions by nature, and in humans, more than 250 proteins are structurally based on A domains. An Avimer consists of a number of different "A domain" monomers (2 - 10) linked by an amino acid linker. Avimers that can bind to a target antigen can be generated using, for example, the methods described in US Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

[0294] Affibody affinity ligands are simple small proteins consisting of a three-helix bundle of a scaffold based on one of the IgG-binding domains of protein A. Protein A is a surface protein from Staphylococcus aureus. The scaffold domain consists of 58 amino acids, of which 13 amino acids are randomized to generate an affibody library with a large number of ligand variants (see, for example, US 5,831,012). Affibody molecules mimic antibodies, and they have a molecular weight of 6 kDa, compared to 150 kDa for antibodies. Despite their small size, the binding sites of affibody molecules are similar to those of antibodies.

[0295] Anticalins are commercially known, for example, Pieris ProteoLab AG. They are derived from lipocalins, a broad class of small and robust proteins that are typically involved in the physiological transport or storage of chemo-sensory or insoluble compounds. Several natural lipocalins are present in human tissues or body fluids. The protein structure resembles that of immunoglobulins, having hypervariable loops at the top of a rigid framework. However, in contrast to antibodies or their recombinant fragments, lipocalins consist of a single polypeptide chain with 160 to 180 amino acid residues, only slightly larger than a single immunoglobulin domain. The combination of four loops forms a binding pocket with significant structural plasticity and the ability to accommodate various side chains. The binding site can thus be reshaped in a proprietary method to recognize defined target molecules of different shapes with high affinity and specificity. One protein of the lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae, has been used to develop anticalins by mutagenizing the combination of four loops. An example of a patent application describing anticalins is PCT Publication No. WO 199916873.

[0296] Affilin molecules are small non-immunoglobulin proteins designed to form specific affinities with proteins and small molecules. New affilin molecules can be rapidly selected from two libraries, each based on a different human-derived scaffold protein. Affilin molecules have no structural homology with immunoglobulin proteins. Currently, two affilin scaffolds are used, one of which is γ-crystallin, a human structural lens protein, and the other is a "ubiquitin"-superfamily protein. Both human scaffolds are very small, show high temperature stability, and are nearly resistant to pH changes and denaturing agents. This high stability is mainly attributed to the extended β-sheet structure of the proteins. Examples of γ-crystallin-derived proteins are described in WO200104144, and examples of "ubiquitin-like" proteins are described in WO2004106368.

[0297] Protein epitope mimetics (PEMs) are medium-sized cyclic peptide-like molecules (MW 1 - 2 kDa) that mimic the β-hairpin secondary structure of proteins, which is the major secondary structure involved in protein-protein interactions.

[0298] Domain antibodies (dAbs) can be used in anti-TCRvb antibody molecules or their multifunctional forms as described herein. They are small functional binding fragments of antibodies corresponding to the variable regions of the antibody heavy or light chains. Domain antibodies are well-expressed in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, for example, U.S. Patent Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patent 0368684 & 0616640; WO05 / 035572, WO04 / 101790, WO04 / 081026, WO04 / 058821, WO04 / 003019, and WO03 / 002609. Nanobodies are derived from the heavy chain of an antibody.

[0299] 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 Nos. 6,765,087, U.S. Patent No. 6,838,254, WO 06 / 079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies can be prepared by removing the hinge region of the IgG4 antibody. Further details of unibodies and methods for their preparation can be found in WO2007 / 059782.

[0300] Anti-TCRVβ Antibody Effector Functions and Fc Variants

[0301] In some embodiments, the anti-TCRVβ antibodies as described herein comprise an Fc region, for example, as described herein. In some embodiments, the Fc region is a wild-type Fc region, such as a wild-type human Fc region. In some embodiments, the Fc region contains variants, such as an Fc region containing at least one amino acid residue addition, substitution, or deletion in the Fc region, which results in, for example, a reduced or eliminated affinity for at least one Fc receptor.

[0302] The Fc region of an antibody interacts with many receptors or ligands, including Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIIA), the complement protein CIq, and other molecules such as protein A and G. These interactions are required for a variety of effector functions and downstream signaling events, including: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0303] In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has a reduced (e.g., eliminated) affinity for Fc receptors (such as the Fc receptors described herein). In some embodiments, the reduced affinity is compared to that of an otherwise similar antibody having a wild-type Fc region.

[0304] In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has one or more of the following characteristics: (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 or all of characteristics (1)-(3) is compared to that of an otherwise similar antibody having a wild-type Fc region.

[0305] In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has a reduced affinity for human Fc receptors such as FcγRI, FcγRII, and / or FcγRIII. In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region comprises a human IgG1 region or a human IgG4 region.

[0306] In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region activates and / or expands T cells, such as described herein. In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has a cytokine profile as described herein, e.g., a cytokine profile different from that of a T cell engager that binds to a receptor or molecule other than the TCRβV region (“non-TCRβV-binding T cell engager”). In some embodiments, a non-TCRβV-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., a CD3ε (CD3e) molecule) or a TCRα (TCRα) molecule.

[0307] 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 content of which is incorporated herein by reference.

[0308] In some embodiments, an anti-TCRVβ antibody as described herein comprises any one or all or any combination of the Fc region variants disclosed in Table 14.

[0309] In some embodiments, an anti-TCRVβ antibody as described herein comprises any one or all or any combination of the Fc region variants (e.g., mutations) disclosed in Table 14. In some embodiments, an anti-TCRVβ antibody as described herein comprises the Asn297Ala (N297A) mutation. In some embodiments, an anti-TCRVβ antibody as described herein comprises the Leu234Ala / Leu235Ala (LALA) mutation.

[0310] Multifunctional molecule

[0311] As used herein, the terms "multifunctional molecule" and "multispecific molecule" are used interchangeably and refer to a molecule having two or more functions (e.g., two or more binding specificities), e.g., a polypeptide. In some embodiments, the functions can include one or more immune cell engagers, one or more tumor-binding molecules, one or more cytokine molecules, one or more matrix modifiers, and other moieties as 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-TCRVβ antibody molecule as described herein.

[0312] In certain embodiments, there is described 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, 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 second portion of the first TCRβV binding portion; (iii) the 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) the fourth polypeptide comprises a second portion of the second TCRβV binding portion; and wherein 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, the fourth polypeptide, or a combination thereof.

[0313] In certain embodiments, described herein is 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, 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 wherein 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.

[0314] In certain embodiments, described herein is 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, 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; wherein 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 wherein the multifunctional polypeptide molecule does not comprise additional TCRβV binding portions other than the first TCRβV binding portion.

[0315] In some embodiments, a first portion of the first TCRβV binding moiety comprises a first variable heavy domain (VH) and a first constant heavy 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, a second portion of the first TCRβV binding moiety comprises a first variable light domain (VL) and a first constant light 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, wherein 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, wherein a 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, a 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.

[0316] 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; (d) 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 (e) a combination thereof.

[0317] 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 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 (b-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; (c-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 (c-2) 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; (d-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 (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 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 (f-1) 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 (f-2) 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.

[0318] In some embodiments, (a-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a functional fragment or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or variant thereof; or a combination thereof; (b-1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a functional fragment or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a functional fragment or variant thereof; or a combination thereof.

[0319] In some embodiments, (1) the N-terminus of the first polypeptide is linked to a first cytokine polypeptide or a functional fragment or variant thereof; the C-terminus of the first polypeptide is linked to a second cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the second polypeptide is linked to a fourth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the third polypeptide is linked to a sixth cytokine polypeptide or a functional fragment or 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 variant thereof; the C-terminus of the fourth polypeptide is linked to an eighth cytokine polypeptide or a functional fragment or variant thereof; or a combination thereof.

[0320] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the second polypeptide, the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the third polypeptide, the seventh cytokine polypeptide, the eighth cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the fourth polypeptide, or a combination thereof.

[0321] In some embodiments, (a) 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; (b) 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; (c) 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; or (d) a combination thereof.

[0322] In some embodiments, (a-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 (a-2) 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; (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 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; or (c-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 (c-2) 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.

[0323] 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.

[0324] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the second polypeptide, the fifth cytokine polypeptide, the sixth cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the third polypeptide, or a combination thereof.

[0325] In some embodiments, the multifunctional polypeptide molecule as 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 the third polypeptide, a linker between at least one cytokine polypeptide or a functional fragment or functional variant thereof and the fourth polypeptide, or a combination thereof.

[0326] In some embodiments, the multifunctional polypeptide molecule as 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 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 wherein the linker is a GS linker. In some embodiments, the linker is a peptide linker, and wherein the linker comprises the sequence of SEQ ID NO:3308 or SEQ ID NO:3643.

[0327] In certain embodiments, there is described herein 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, 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; (iii) the 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) the fourth polypeptide comprises a second portion of the second TCRβV binding portion; and wherein the first cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the C-terminus of the second polypeptide, and the second cytokine polypeptide or a functional fragment or functional variant thereof is covalently linked to the C-terminus of the fourth polypeptide.

[0328] In certain embodiments, described herein 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, 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 second portion of the first TCRβV binding moiety; (iii) the third polypeptide comprises a first portion of a second TCRβV binding moiety and a second dimerization module linked to the first portion of the second TCRβV binding moiety; and (iv) the fourth polypeptide comprises a second portion of the second TCRβV binding moiety; and wherein the 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.

[0329] In certain embodiments, described herein 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, 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 second portion of the first TCRβV binding moiety; (iii) the third polypeptide comprises a first portion of a second TCRβV binding moiety and a second dimerization module linked to the first portion of the second TCRβV binding moiety; and (iv) the fourth polypeptide comprises a second portion of the second TCRβV binding moiety; and wherein the 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.

[0330] In certain embodiments, described herein is 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, 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 second portion of the first TCRβV binding moiety; and (iii) the third polypeptide comprises a second dimerization module; wherein the 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 wherein the multifunctional polypeptide molecule does not comprise an additional TCRβV binding moiety other than the first TCRβV binding moiety.

[0331] In some embodiments, a first portion of the first TCRβV binding moiety 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.

[0332] In some embodiments, a second portion of the first TCRβV binding moiety comprises a first VL and a first CL linked to the first VL. In some embodiments, the first CL is linked to the C-terminus of the first VL.

[0333] 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, a 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, a 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.

[0334] In some embodiments, the multifunctional polypeptide molecule as described herein further comprises a linker between the first part of the first TCRβV binding portion and the first dimerization module, a linker between the first part of the second TCRβV binding portion 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 the third polypeptide, a linker between at least one cytokine polypeptide or a functional fragment or functional variant thereof and the fourth polypeptide, or a combination thereof. In some embodiments, the multifunctional polypeptide molecule as described herein further comprises a linker between the first part 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 third polypeptide, or a combination thereof. In some embodiments, the linker is selected from cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. In some embodiments, the linker is a peptide linker, and wherein the linker is a GS linker. In some embodiments, the linker is a peptide linker, and wherein the linker comprises the sequence of SEQ ID NO: 3308 or SEQ ID NO: 3643.

[0335] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises any one selected from Fab, F(ab’)2, Fv, single-chain Fv (scFv), single-domain antibodies, diabodies (dAb), camel antibodies, and combinations thereof. In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises scFv or Fab.

[0336] In some embodiments, the multifunctional polypeptide molecule does not comprise an additional antigen-binding portion other than the TCRβV binding portion. In some embodiments, the multifunctional polypeptide molecule further comprises an additional antigen-binding portion that is not the TCRβV binding portion.

[0337] In certain embodiments, described herein is a multifunctional polypeptide molecule that comprises 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, wherein: (i) the first polypeptide comprises a first TCRβV binding portion and a first dimerization module linked to the C-terminus of the first TCRβV binding portion, wherein the first TCRβV binding portion comprises a first VL and a first VH; and (ii) the second polypeptide comprises a second TCRβV binding portion and a second dimerization module linked to the C-terminus of the second TCRβV binding portion; wherein the 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; wherein the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises an scFv; and wherein the multifunctional polypeptide molecule does not comprise additional antigen-binding portions other than the first TCRβV binding portion and the second TCRβV binding portion.

[0338] In certain embodiments, described herein is a multifunctional polypeptide molecule that comprises 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, wherein: (i) the first polypeptide comprises a first TCRβV binding portion and a first dimerization module linked to the C-terminus of the first TCRβV binding portion, wherein the first TCRβV binding portion comprises a first VL and a first VH; and (ii) the second polypeptide comprises a second dimerization module; wherein the 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; wherein the first TCRβV binding portion comprises an scFv; wherein the multifunctional polypeptide molecule does not comprise additional antigen-binding portions other than the first TCRβV binding portion; and wherein the multifunctional polypeptide molecule does not comprise additional TCRβV binding portions other than the first TCRβV binding portion.

[0339] 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 (c) a combination thereof.

[0340] In some embodiments, the first cytokine polypeptide, the second cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the first polypeptide, the third cytokine polypeptide, the fourth cytokine polypeptide, or a combination thereof is within a single continuous polypeptide chain of the second polypeptide, or a combination thereof.

[0341] In some embodiments, the multifunctional polypeptide molecule as described herein further comprises a linker between the first TCRβV binding portion and the first dimerization module, a linker between the second TCRβV binding portion 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.

[0342] In some embodiments, the multifunctional polypeptide molecule as 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 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. In some embodiments, the linker is selected from cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. In some embodiments, the linker is a peptide linker, and wherein the linker is a GS linker. In some embodiments, the linker is a peptide linker, and wherein the linker comprises the sequence of SEQ ID NO:3308 or SEQ ID NO:3643.

[0343] In some embodiments, the multifunctional polypeptide molecule comprises at least two cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least three cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least four cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least five cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least six cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least seven cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises at least eight cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises two cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises three cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises four cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises five cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises six cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises seven cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises eight cytokine polypeptides. In some embodiments, the multifunctional polypeptide molecule comprises two cytokine polypeptides, each of which is separately 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 polypeptide and a third polypeptide; a second polypeptide and a fourth polypeptide; or a third polypeptide and a fourth polypeptide. In some embodiments, the multifunctional polypeptide molecule comprises three cytokine polypeptides, each of which is separately 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. In some embodiments, the multifunctional polypeptide molecule comprises four cytokine polypeptides, each of which is separately linked to a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. In some embodiments, the cytokine polypeptide is not linked to a polypeptide comprising a first TCRβV binding portion.

[0344] In some embodiments, at least one cytokine polypeptide is selected from interleukin 2 (IL-2) or a fragment or functional fragment or functional variant thereof, or a combination thereof.

[0345] 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 the sequence of SEQ ID NO: 2191. In some embodiments, the sequence of at least one cytokine polypeptide 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: 2191. In some embodiments, the sequence of at least one cytokine polypeptide is the sequence of SEQ ID NO: 2191.

[0346] In some embodiments, a 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, a 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 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 sequence of the variant is the sequence of SEQ ID NO: 2270.

[0347] 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.

[0348] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from an IgG1 Fc region or a fragment thereof, an IgG2 Fc region or a fragment thereof, an IgG3 Fc region or a fragment thereof, an IgGA1 Fc region or a fragment thereof, an IgGA2 Fc region or a fragment thereof, an IgG4 Fc region or a fragment thereof, an IgJ Fc region or a fragment thereof, an IgM Fc region or a fragment thereof, an IgD Fc region or a fragment thereof, and an IgE Fc region or a fragment thereof.

[0349] In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from a human IgG1 Fc region or a fragment thereof, a human IgG2 Fc region or a fragment thereof, and a human IgG4 Fc region or a fragment thereof.

[0350] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises an Fc interface having one or more of paired cavity-protrusion, electrostatic interaction, or chain exchange, wherein dimerization of the first Fc region and the second Fc region is enhanced, as indicated by a greater hetero-oligomer:homo-oligomer form ratio relative to dimerization of Fc regions having a non-engineered interface. In some embodiments, dimerization of the first Fc region and the second Fc region is enhanced 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, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 250-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, or 10000-fold relative to dimerization of Fc regions having a non-engineered interface. In some embodiments, dimerization of the first Fc region and the second Fc region is enhanced 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, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 250-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, or 10000-fold relative to dimerization of Fc regions having a non-engineered interface.In some embodiments, the dimerization of the first Fc region and the second Fc region is enhanced 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-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 250-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold or 10000-fold relative to the dimerization of an Fc region with an unengineered interface.

[0351] In some embodiments, the first Fc region, the second Fc region, or a combination thereof comprises the amino acid substitutions listed in Table 14.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof binds to one or more of the TCRβV subfamilies selected from: (i) the TCRβV6 subfamily, which includes one or more selected from TCRβV6-4*01, TCRβV6-4*02, TCRβV6-9*01, TCRβV6-8*01, TCRβV6-5*01, TCRβV6-6*02, TCRβV6-6*01, TCRβV6-2*01, TCRβV6-3*01, and TCRβV6-1*01, and (ii) the TCRβV10 subfamily, which includes one or more selected from TCRβV10-1*01, TCRβV10-1*02, TCRβV10-3*01, and TCRβV10-2*01.

[0356] In some embodiments, the first TCRβV binding portion and the second TCRβV binding portion are the same. In some embodiments, the first TCRβV binding portion and the second TCRβV binding portion are different.

[0357] In some embodiments, the first TCRβV binding portion and the second TCRβV binding portion each bind to one or more members of the TCRβV6 subfamily and one or more members of the TCRβV10 subfamily, respectively.

[0358] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises: (i) HC CDR1, HC CDR2, and HC CDR3 having amino acid sequences with at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; (ii) LC CDR1, LC CDR2, and LC CDR3 having amino acid sequences with at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; or (iii) a combination thereof. In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises: (i) HC CDR1, HC CDR2, and HC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; (ii) LC CDR1, LC CDR2, and LC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; or (iii) a combination thereof.

[0359] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises: (i) HC CDR1, HC CDR2, and HC CDR3 having amino acid sequences with at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity, respectively, to any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; (ii) LC CDR1, LC CDR2, and LC CDR3 having amino acid sequences with at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity, respectively, to any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1; or (iii) a combination thereof. In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises: (i) HC CDR1, HC CDR2, and HC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1, respectively; (ii) LC CDR1, LC CDR2, and LC CDR3 having any one of the CDR1, CDR2, and CDR3 sequences listed in Table 1, respectively; or (iii) a combination thereof.

[0360] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises: (i) a VH that comprises framework regions (FRs) that comprise framework 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4; (ii) a VL that comprises FRs that comprise FR1, FR2, FR3, and FR4 having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine 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 that comprises FRs that comprise FR1, FR2, FR3, and FR4 having the sequences of non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4; (ii) a VL that comprises FRs that comprise FR1, FR2, FR3, and FR4 having the sequences of non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4; or (iii) a combination thereof.

[0361] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises: (i) a VH that comprises FR1, FR2, FR3, and FR4 having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4; (ii) a VL that comprises an FR that comprises FR1, FR2, FR3, and FR4 having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4, respectively; or (iii) a combination thereof. In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises: (i) a VH that comprises an FR that comprises FR1, FR2, FR3, and FR4 having the sequences of non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4, respectively; (ii) a VL that comprises an FR that comprises FR1, FR2, FR3, and FR4 having the sequences of non-murine germline FR1, non-murine germline FR2, non-murine germline FR3, and non-murine germline FR4, respectively; or (iii) a combination thereof.

[0362] In some embodiments, the VH comprises an FR3 that comprises (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 that comprises a phenylalanine at position 10 according to Kabat numbering. In some embodiments, the VL comprises an FR2 that comprises (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 that comprises a phenylalanine at position 87 according to Kabat numbering.

[0363] 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 a sequence with at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any 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 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, the sequence of which is a sequence with at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to any 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 of the heavy chain constant region 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 the heavy chain constant region of IgM or a fragment thereof. In some embodiments, the heavy chain constant region of IgM comprises a sequence with 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 heavy chain constant region of IgM comprises the sequence of SEQ ID NO:73. In some embodiments, the sequence of the heavy chain constant region of IgM is the sequence of SEQ ID NO:73.

[0364] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises the heavy chain constant region of IgJ or a fragment thereof. In some embodiments, the heavy chain constant region of IgJ comprises a sequence with 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 heavy chain constant region of IgJ comprises the sequence of SEQ ID NO:76. In some embodiments, the sequence of the heavy chain constant region of IgJ is the sequence of SEQ ID NO:76.

[0365] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises the 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.

[0366] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises the 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.

[0367] In some embodiments, the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, or a combination thereof comprises the heavy chain constant region of IgG1 or a fragment thereof. In some embodiments, the heavy chain constant region of IgG1 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 heavy chain constant region of IgG1 comprises the sequence of SEQ ID NO:41. In some embodiments, the sequence of the heavy chain constant region of IgG1 is the sequence of SEQ ID NO:41. In some embodiments, the heavy chain constant region of IgG1 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 heavy chain constant region of IgG1 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.

[0368] 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 with 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 light 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 light chain constant region having any one of the light chain constant region sequences listed in Table 3, or a combination thereof.

[0369] 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 the κ chain or a fragment thereof. In some embodiments, the light chain constant region of the κ chain comprises the light chain constant region sequences listed in Table 3.

[0370] In some embodiments, the light chain constant region of the κ chain comprises a sequence with 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 κ 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 κ chain is a sequence with 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 κ chain is the sequence of SEQ ID NO:39 or SEQ ID NO:3644.

[0371] In some embodiments, the first TCRβV binding moiety, the second TCRβV binding moiety, or a combination thereof comprises a light chain that comprises FR1, and FR1 comprises: (i) aspartic acid at position 1 according to Kabat numbering; (ii) asparagine at position 2 according to Kabat numbering; (iii) leucine at position 4 according to Kabat numbering; or (iv) a combination thereof.

[0372] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof comprises a light chain, the light chain comprising FR3, the FR3 comprising: (i) glycine at position 66 according to Kabat numbering; (ii) asparagine at position 69 according to Kabat numbering; (iii) tyrosine at position 71 according to Kabat numbering; or (iv) a combination thereof.

[0373] In some embodiments, the first TCRβV binding portion, the second TCRβV binding portion, or a combination thereof binds to an outward-facing region on the TCRβV protein. In some embodiments, the outward-facing region on the TCRβV protein comprises a structurally conserved region of TCRβV, the structurally conserved region having a similar structure in one or more TCRβV subfamilies.

[0374] Cytokine molecule

[0375] In some embodiments, the multifunctional molecule comprises a cytokine molecule. As used herein, "cytokine molecule" or "cytokine polypeptide", which may be used interchangeably herein, refers to the full-length, fragment, or variant of a cytokine; a cytokine further comprising a receptor domain such as a cytokine receptor dimerization domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) against a cytokine receptor, which elicits 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. The cytokine molecule can be monomeric or dimeric. In an embodiment, the cytokine molecule can further comprise a cytokine receptor dimerization domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor.

[0376] Cytokines are generally polypeptides that affect cell activity, for example, through signal transduction pathways. Thus, cytokines of multispecific or multifunctional polypeptides are useful and can be associated with receptor-mediated signal transduction that transmits signals from outside the cell membrane to regulate intracellular responses. Cytokines are protein signaling compounds that are mediators of the immune response. They control many different cell functions, including proliferation, differentiation, and cell survival / apoptosis; cytokines are also involved in a variety of pathophysiological processes, including viral infections and autoimmune diseases. Cytokines are synthesized upon different stimulation of various cells of the innate immune system (monocytes, macrophages, dendritic cells) and the adaptive immune system (T cells and B cells). Cytokines can be divided into two groups: pro-inflammatory and anti-inflammatory. Pro-inflammatory cytokines, including IFNγ, IL-1, IL-6, and TNF-α, are mainly derived from innate immune cells and Th1 cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL-13, and IL-5, are synthesized by Th2 immune cells.

[0377] The present disclosure particularly provides multi-specific (e.g., bispecific, trispecific, tetra-specific) or multi-functional molecules that include, for example, engineered to include one or more cytokine molecules, such as immunomodulatory (e.g., pro-inflammatory) cytokines and variants thereof, such as functional variants thereof. Thus, in some embodiments, the cytokine molecule is an interleukin or a variant thereof, such as 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.

[0378] In certain embodiments, the cytokine is a single-chain cytokine. In certain embodiments, the cytokine is a multi-chain cytokine (e.g., the cytokine comprises two or more (e.g., two) polypeptide chains.

[0379] Examples of useful cytokines include, but are not limited to, IL-2. In some embodiments, the cytokine of the multi-specific or multi-functional polypeptide is IL-2. In certain embodiments, the cytokine is mutated to remove N- and / or O-glycosylation sites. Elimination of glycosylation increases the homogeneity of the product obtainable in recombinant production.

[0380] In some embodiments, the cytokine of the multispecific or multifunctional polypeptide is IL-2. In a specific embodiment, the IL-2 cytokine can elicit one or more of the following cellular responses: proliferation of activated T lymphocytes, differentiation of activated T lymphocytes, cytotoxic T lymphocyte (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 that has a reduced binding affinity for the α subunit of the IL-2 receptor. The α subunit (also known as CD 25) forms a heterotrimeric high-affinity IL-2 receptor together with the β subunit and the γ subunit (known as CD 122 and CD132, respectively), while the dimer receptor consisting only of the β and γ subunits is called the intermediate-affinity IL-2 receptor. As described in PCT patent application number PCT / EP2012 / 051991 (which is incorporated herein by reference in its entirety), compared with the wild-type IL-2 polypeptide, the mutant IL-2 polypeptide with reduced binding to the α subunit of the IL-2 receptor has a reduced ability to induce IL-2 signaling in regulatory T cells, induces less activation-induced cell death (AICD) in T cells, and has a reduced toxicity profile in vivo. The use of such a cytokine with reduced toxicity is particularly advantageous in the multispecific or multifunctional polypeptides of the present invention, which have a longer serum half-life due to the presence of an Fc domain. In some embodiments, the mutant IL-2 cytokine of the multispecific or multifunctional polypeptide according to the present invention comprises at least one amino acid mutation that reduces or eliminates the affinity of the mutant IL-2 cytokine for the α subunit (CD25) of the IL-2 receptor compared to the unmutated IL-2 cytokine, but retains the affinity of the mutant IL-2 cytokine for the intermediate-affinity IL-2 receptor (composed 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 a specific embodiment, the mutant IL-2 cytokine contains one, two, or three amino acid substitutions at one, two, or three positions selected from the positions corresponding to residues 42, 45, and 72 of human IL-2. In a more specific embodiment, the mutant IL-2 cytokine contains three amino acid substitutions at the positions corresponding to residues 42, 45, and 72 of human IL-2. In an even more specific embodiment, the mutant IL-2 cytokine is human IL-2, which comprises the amino acid substitutions F42A, Y45A, and L72G. In some embodiments, the mutant IL-2 cytokine additionally contains an amino acid mutation at the position corresponding to position 3 of human IL-2, which eliminates the O-glycosylation site of IL-2.Specifically, said additional amino acid mutations are amino acid substitutions in which threonine residues are replaced by alanine residues. Specific mutant IL-2 cytokines useful in the present invention contain four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. The specific amino acid substitutions are T3A, F42A, Y45A and L72G. As shown in PCT patent application No. PCT / EP2012 / 051991 and the appended examples, said quadruple mutant IL-2 polypeptide (IL-2qm) has no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in Treg cells, and reduced in vivo toxicity profile. However, it retains the ability to activate IL-2 signaling in effector cells, induce proliferation of effector cells, and produce IFN-γ as a secondary cytokine by NK cells.

[0381] The IL-2 or mutant IL-2 cytokine according to any one of the above embodiments may contain other mutations that provide further advantages such as increased expression or stability. For example, the cysteine at position 125 can be replaced with a neutral amino acid such as alanine to avoid formation of disulfide-bridged IL-2 dimers. Thus, in certain embodiments, the IL-2 or mutant IL-2 cytokine of the multispecific or multifunctional polypeptide of the present invention contains an additional amino acid mutation at the position corresponding to residue 125 of human IL-2. In some embodiments, said additional amino acid mutation is the amino acid substitution C125A.

[0382] In a specific embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide comprises SEQ ID NO: 2270

[0383] [APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT] polypeptide sequence.

[0384] In another specific embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide comprises SEQ ID NO: 2280

[0385] The polypeptide sequence of [[APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT]].

[0386] Mutant cytokine molecules that can be used 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 coding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing. Substitutions or insertions can involve natural as well as unnatural amino acid residues. Amino acid modifications include well-known chemical modification methods, such as addition or removal of glycosylation sites or carbohydrate attachment, etc.

[0387] In some embodiments, the multispecific or multifunctional polypeptides of the invention bind to cytokine receptors with a dissociation constant (K D ) that is 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 greater than that of a control cytokine. In another embodiment, the multispecific or multifunctional polypeptide binds to cytokine receptors with a K D that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than that of a corresponding multispecific or multifunctional polypeptide comprising two or more effector moieties. In another embodiment, the multispecific or multifunctional polypeptide binds to cytokine receptors with a dissociation constant K D that is approximately 10 times greater than that of a corresponding multispecific or multifunctional polypeptide comprising two or more cytokines.

[0388] In some embodiments, the multispecific molecules as described herein include cytokine molecules. In an embodiment, the cytokine molecule includes the 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 (e.g., an agonistic antibody) against a cytokine receptor.

[0389] In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2, e.g., comprising the amino acid sequence:

[0390] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:2191), a fragment thereof, or an amino acid sequence that is substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid change to the amino acid sequence of SEQ ID NO:2191, but not more than five, ten, or fifteen changes (e.g., substitutions, deletions, or insertions, such as conservative substitutions)).

[0391] Immune cell linker

[0392] In some embodiments, the multifunctional molecule further comprises an immune cell linker. An "immune cell linker" refers to one or more binding specificities that bind 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 macrophages. The immune cell linker 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, B cell antigen, dendritic cell antigen, and / or macrophage antigen). In embodiments, the immune cell linker specifically binds to a target immune cell, e.g., preferentially binds to a target immune cell. For example, when the immune cell linker is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, NK cell antigen, B cell antigen, dendritic cell antigen, and / or macrophage antigen) with a dissociation constant of less than about 10 nM.

[0393] The immune cell linker of the multispecific or multifunctional molecule as described herein, e.g., the first and / or second immune cell linker, can mediate binding and / or activation with immune cells such as immune effector cells. In some embodiments, the immune cells are selected from T cells, NK cells, B cells, dendritic cells, or macrophage linkers, or combinations thereof. In some embodiments, the immune cell linker is selected from one, two, three, or all of a T cell linker, an NK cell linker, a B cell linker, a dendritic cell linker, or a macrophage linker, or combinations thereof. The immune cell linker can be an agonist of the immune system. In some embodiments, the immune cell linker can be an antibody molecule, a ligand molecule (e.g., a ligand further comprising an immunoglobulin constant region, such as an Fc region), a small molecule, a nucleotide molecule.

[0394] Antibody molecule

[0395] In some embodiments, the antibody molecule binds to a cancer antigen, e.g., a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, e.g., a mammalian, e.g., a human cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, e.g., a mammalian, e.g., a human immune cell antigen. For example, the antibody molecule specifically binds to an epitope on a cancer antigen or an immune cell antigen, e.g., a linear or conformational epitope.

[0396] In some embodiments, the antibody molecule is a monospecific antibody molecule and binds to a single epitope. For example, a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each sequence binding to the same epitope.

[0397] In some embodiments, the antibody molecule is a multispecific or multifunctional antibody molecule, e.g., it comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the multiple has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the multiple has binding specificity for a second epitope. In some embodiments, the first epitope and the second epitope are on the same antigen, e.g., the same protein (or a subunit of a multimeric protein). In some embodiments, the first epitope and the second epitope overlap. In some embodiments, the first epitope and the second epitope do not overlap. In some embodiments, the first epitope and the second epitope are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, the multifunctional antibody molecule comprises third, fourth or fifth immunoglobulin variable domains. In some embodiments, the multifunctional antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule or a tetra - specific antibody molecule.

[0398] In some embodiments, the multifunctional antibody molecule is a bispecific antibody molecule. The bispecific antibody is specific for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In some embodiments, the first epitope and the second epitope are on the same antigen, such as the same protein (or subunit of a multimeric protein). In some embodiments, the first epitope and the second epitope overlap. In some embodiments, the first epitope and the second epitope do not overlap. In some embodiments, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In some embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecule comprises a half-antibody having binding specificity for a first epitope and a half-antibody having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecule comprises a half-antibody or a fragment thereof having binding specificity for a first epitope and a half-antibody or a fragment thereof having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecule comprises a scFv or Fab or a fragment thereof having binding specificity for a first epitope, and a scFv or Fab or a fragment thereof having binding specificity for a second epitope.

[0399] In some embodiments, the antibody molecule comprises diabodies, single-chain molecules, and antigen-binding fragments of antibodies (e.g., Fab, F(ab’)2, and Fv). For example, the antibody molecule may comprise heavy (H) chain variable domain sequences (abbreviated herein as VH) and light (L) chain variable domain sequences (abbreviated herein as VL). In some embodiments, the antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half-antibody). In another example, the antibody molecule includes 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 modifying intact antibodies or synthesized de novo using recombinant DNA techniques. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and any subclass of antibodies (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. The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, κ or λ. The term “immunoglobulin” (Ig) can be used interchangeably with the term “antibody” herein.

[0400] Examples of antigen-binding fragments of antibody molecules include: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VL and VH domains of a single arm of an antibody; (v) diabody (dAb) fragment consisting of VH domains; (vi) camelid or camelized variable domains; (vii) 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); (viii) single domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as intact antibodies.

[0401] Antibody molecules include intact molecules and functional fragments thereof. The constant region of an antibody molecule can be altered, for example by mutagenesis, to modify the properties of the antibody (e.g., increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function).

[0402] An antibody molecule can also be a single-domain antibody. A single-domain antibody can include an antibody in which its complementarity-determining region is part of a single-domain polypeptide. Examples include but are not limited to heavy-chain antibodies, antibodies that are naturally devoid of light chains, single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain scaffolds not derived from antibodies. A single-domain antibody can be any single-domain antibody in the art, or any future single-domain antibody. A single-domain antibody can be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the invention, a single-domain antibody is a naturally occurring single-domain antibody called a heavy-chain antibody lacking a light chain. Such single-domain antibodies are disclosed, for example, in WO 9404678. For clarity, the variable domain derived from such a heavy-chain antibody that is naturally lacking a light chain is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camel, llama, dromedary, alpaca, and guanaco). In addition to camelids, other species may also produce heavy-chain antibodies that are naturally lacking a light chain; such VHHs are within the scope of the present invention.

[0403] The VH and VL regions can be subdivided into hypervariable regions, called "complementarity-determining regions" (CDRs), interspersed with more conserved regions, called "framework regions" (FR or FW).

[0404] The framework regions and the CDRs have been precisely defined by a variety of methods (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department 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. See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In Antibody Engineering Lab Manual (editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0405] As used herein, the terms "complementary determining region" and "CDR" refer to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0406] The precise amino acid sequence boundaries of a given CDR can be determined using any of a variety of known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme are sometimes also referred to as "hypervariable loops."

[0407] For example, according to Kabat, the CDR amino acid residue numbers in the heavy chain variable domain (VH) are 31 - 35 (HCDR1), 50 - 65 (HCDR2), and 95 - 102 (HCDR3); while the CDR amino acid residue numbers in the light chain variable domain (VL) are 24 - 34 (LCDR1), 50 - 56 (LCDR2), and 89 - 97 (LCDR3). According to Chothia, the CDR amino acid numbers in VH are 26 - 32 (HCDR1), 52 - 56 (HCDR2), and 95 - 102 (HCDR3); while the amino acid residue numbers in VL are 26 - 32 (LCDR1), 50 - 52 (LCDR2), and 91 - 96 (LCDR3).

[0408] Each VH and VL generally includes three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0409] The antibody molecule can be a polyclonal or monoclonal antibody.

[0410] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules consisting of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope. Monoclonal antibodies can be prepared by hybridoma technology or by methods that do not use hybridoma technology (such as recombinant methods).

[0411] Antibodies can be produced recombinantly, for example, by phage display or by combinatorial methods or by yeast display.

[0412] Phage display and combinatorial methods for generating antibodies are known in the art (as described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; Fuchs et 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; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins et 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., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, the entire contents of which are incorporated herein by reference).

[0413] Yeast display methods for generating or identifying antibodies are known in the art, e.g., as described in Chao et al. (2006) Nature Protocols 1(2):755-68, the entire contents of which are incorporated herein by reference.

[0414] In some embodiments, the antibody is a fully human antibody (e.g., an antibody prepared in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as an antibody of a rodent (mouse or rat), goat, primate (e.g., monkey), camel. Preferably, the non-human antibody is a rodent antibody (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0415] Transgenic mice carrying human immunoglobulin genes rather than mouse systems can be used to produce human monoclonal antibodies. Spleen cells from these transgenic mice immunized with the antigen of interest are used to generate hybridomas that secrete human mAbs with specific affinity for epitopes of human proteins (see, for example, Wood et al., International Application WO 91 / 00906, Kucherlapati et al., PCT Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N et al 1994 Nature 368:856-859; Green, L.L. et al 1994 Nature Genet. 7:13-21; Morrison, S.L. 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).

[0416] Antibody molecules can be antibody molecules that produce variable regions or portions thereof, such as CDRs, in non-human organisms such as rats or mice. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. Antibody molecules produced in non-human organisms such as rats or mice and then modified, for example, in the variable framework or constant region to reduce antigenicity in humans are within the scope of the present invention.

[0417] An "effectively human" protein is a protein that essentially does not elicit a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. In many cases, for example, if antibody molecules are administered repeatedly, such as for the treatment of chronic or recurrent diseases, HAMA can be a problem. HAMA responses can potentially render repeated antibody administration ineffective due to increased clearance of the antibody from the serum (see, for example, Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)), and also due to potential allergic reactions (see, for example, LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0418] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 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 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).

[0419] A humanized or CDR-grafted antibody will have at least one or two, but typically all three, recipient CDRs (of the immunoglobulin heavy and / or light chain) replaced by donor CDRs. The antibody can be replaced by at least a portion of non-human CDRs, or only some CDRs can be replaced by non-human CDRs. Only the number of CDRs required for antigen binding needs to be replaced. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the recipient will be a human framework or a 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 "recipient". In some embodiments, the donor immunoglobulin is non-human (e.g., rodent). The recipient framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence having about 85% or higher, preferably 90%, 95%, 99% or higher identity thereto.

[0420] As used herein, the term "consensus sequence" refers to the sequence formed by the most frequently occurring amino acids (or nucleotides) in a related family of 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 may be included in the consensus sequence. A "consensus framework" refers to the framework regions in a consensus immunoglobulin sequence.

[0421] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, S.L., 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214 and Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the entire contents of which are incorporated herein by reference).

[0422] Humanized or CDR-grafted antibody molecules can be produced by CDR grafting or CDR replacement, in which one, two or all of the CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Patent 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. 5,225,539, the entire contents of which are expressly incorporated herein by reference. Winter describes CDR grafting methods useful for preparing the humanized antibodies of the present invention (British Patent Application GB 2188638A filed Mar. 26, 1987; Winter 5,225,539), the contents of which are expressly incorporated herein by reference.

[0423] Humanized antibody molecules are also within the scope of the present invention in which specific amino acids have been replaced, deleted or added. The criteria for selecting amino acids from the donor are described in U.S. 5,585,089, e.g., columns 12-16 of U.S. 5,585,089, e.g., columns 12-16 of U.S. 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596A1 published Dec. 23, 1992.

[0424] The antibody molecule can be a single-chain antibody. The single-chain antibody (scFv) can be engineered (see, e.g., Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) ClinCancer Res 2:245-52). The single-chain antibody can be dimerized or multimerized to produce multivalent antibodies specific for different epitopes of the same target protein.

[0425] In still other embodiments, the antibody molecule has a heavy-chain constant region selected from the heavy-chain constant regions of, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly selected from the (e.g., human) heavy-chain constant regions of, for example, IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light-chain constant region selected from the (e.g., human) light-chain constant regions of, for example, κ or λ. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, 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 a reduced ability to bind Fc receptors or no ability to bind Fc receptors. For example, it is an isotype or subtype, fragment, or other mutant that does not support binding to Fc receptors, e.g., one having a mutagenized or deleted Fc receptor-binding region.

[0426] Methods for altering the antibody constant region are known in the art. Antibodies with altered functions (e.g., 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, e.g., EP 388,151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, the entire contents of which are incorporated herein by reference). Similar types of alterations can be described that, if applied to murine or other species, would reduce or eliminate these functions in immunoglobulins.

[0427] Antibody molecules can be derivatized or conjugated to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is an antibody molecule that has been modified. Methods of derivatization include, but are not limited to, adding a fluorescent moiety, a radiolabeled nucleotide, a toxin, an enzyme, or an affinity ligand such as biotin. Accordingly, the antibody molecules of the invention are intended to include derivatized forms and other modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0428] One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (of the same type or different types, e.g., to produce a bispecific antibody). Suitable cross-linking agents include heterobifunctional cross-linking agents having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0429] CDR-grafted scaffolds

[0430] In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, e.g., a fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to the fold of the smallest functional antibody fragment (the variable domain of the 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 has no disulfide bonds; thus, unlike antibodies and their fragments, Fn3 is stable under reducing conditions (see, e.g., 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, e.g., to select a domain that binds to an antigen / marker / cell described herein.

[0431] In some embodiments, the scaffold domain, such as a fold domain, is antibody-based, such as a "minibody" scaffold generated by deleting three β-strands from the heavy chain variable domain of 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). The "minibody" can be used to present two hypervariable loops. In some embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al. WO99 / 45110) or a domain derived from tendamistatin, a 74-residue six-stranded β-sheet sandwich structure 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, such as to select a domain that binds to a marker / antigen / cell described herein. Another exemplary scaffold domain is a β-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00 / 60070).

[0432] Other exemplary scaffold 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 domain, colicin, BPTI, etc.); TPR repeats; the trefoil structure; zinc finger domains; DNA-binding proteins; particularly monomeric DNA-binding proteins; RNA-binding proteins; enzymes, such as proteases (particularly inactivated proteases), ribonucleases; chaperone molecules, such as thioredoxin and heat shock proteins; and intracellular signaling domains (such as SH2 and SH3 domains). See, e.g., US20040009530 and US 7,501,121, which are incorporated herein by reference.

[0433] In some embodiments, the scaffold domain is evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) three-dimensional structure, and / or (4) stability data within ranges of pH, temperature, salinity, organic solvents, oxidant concentration. In some embodiments, the scaffold domain is a small, stable protein domain, such as a protein of fewer than 100, 70, 50, 40, or 30 amino acids. The domain can include one or more disulfide bonds or can chelate a metal, such as zinc.

[0434] Antibody-based fusion

[0435] Multiple forms can be generated that contain additional binding entities attached to the N- or C-terminus of an antibody. These fusions with single-chain or disulfide-stabilized Fv or Fab result in the production of tetravalent molecules with bivalent binding specificity for each antigen. Combinations of scFv and scFab with IgG can produce molecules that recognize three or more different antigens.

[0436] Antibody-Fab fusion

[0437] 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. Typically, the antibody and the Fab will have a common light chain. The antibody fusion can be produced by (1) engineering the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell for expression of the fusion protein. As described by Coloma, J. et al. (1997) Nature Biotech 15:159, it appears that antibody-scFv fusions can be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0438] Antibody-scFv fusion

[0439] An antibody-scFv fusion is a bispecific antibody that contains a conventional antibody fused to the C-terminus of the antibody heavy chain and an scFv with a unique specificity. The scFv can be fused directly to the C-terminus via the scFv heavy chain or via a linker peptide. The antibody fusion can be produced by (1) engineering the DNA sequence of the target fusion and (2) transfecting the target DNA into a suitable host cell for expression of the fusion protein. As described by Coloma, J. et al. (1997) Nature Biotech 15:159, it appears that antibody-scFv fusions can be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv.

[0440] Variable domain immunoglobulin DVD

[0441] A related form is the dual variable domain immunoglobulin (DVD), which consists of VH and VL domains at a second specific position at the N-terminus of the V domain, linked by a shorter linker sequence.

[0442] Other exemplary multifunctional antibody forms include, for example, those described in the following patents: US20160114057A1, US20130243775A1, US20140051833, US20130022601, US20150017187A1, US20120201746A1, US20150133638A1, US20130266568A1, US20160145340A1, WO2015127158A1, US20150203591A1, US20140322221A1, US20130303396A1, US20110293613, US20130017200A1, US20160102135A1, WO2015197598A2, WO2015197582A1, US9359437, US20150018529, WO2016115274A1, WO2016087416A1, US20080069820A1, US9145588B, US7919257, and US20150232560A1. Exemplary multifunctional molecules in the full antibody-Fab / scFab form include those described in the following patents: US9382323B2, US20140072581A1, US20140308285A1, US20130165638A1, US20130267686A1, US20140377269A1, US7741446B2, and WO1995009917A1. Exemplary multifunctional molecules in the domain-swapped form include those described in the following patents: US20150315296A1, WO2016087650A1, US20160075785A1, WO2016016299A1, US20160130347A1, US20150166670, US8703132B2, US20100316645, US8227577B2, US20130078249.

[0443] Fc-containing multifunctional molecules

[0444] In some embodiments, the multifunctional molecules as 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 specifically, the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0445] In some embodiments, the constant region of an immunoglobulin chain (e.g., the Fc region) is altered, e.g., mutated, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0446] In other embodiments, the interface between a first immunoglobulin chain constant region and a second immunoglobulin chain constant region (e.g., a first Fc region and a second Fc region) is altered (e.g., mutated) to increase or decrease dimerization, e.g., relative to a non-engineered interface (e.g., a naturally occurring interface). For example, dimerization of an immunoglobulin chain constant region (e.g., the Fc region) can be enhanced by providing one or more of the following for the Fc interface of the first Fc region and the second Fc region: paired protrusion-cavity ("knob-into-hole structure"), electrostatic interactions, or chain exchange, such that, e.g., a greater ratio of heteromultimers to homomultimers is formed relative to the non-engineered interface.

[0447] In some embodiments, the multifunctional molecule comprises paired amino acid substitutions at one or more positions selected from, e.g., 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of, e.g., human IgG1. For example, the constant region of an immunoglobulin chain (e.g., the Fc region) can comprise paired amino acid substitutions selected from: T366S, L368A, or Y407V (e.g., corresponding to the cavity or hole) and T366W (e.g., corresponding to the protrusion or knob).

[0448] In other embodiments, the multifunctional molecule comprises a half-life extender, e.g., human serum albumin, or an antibody molecule against human serum albumin.

[0449] In some embodiments, the Fc comprises the exemplary Fc modifications listed in Table 14. Heterodimeric antibody molecules and methods of making the same

[0450] A variety of methods for generating multifunctional antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multifunctional antibody formats and methods of making the multifunctional antibodies are also disclosed in, e.g., 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 are incorporated herein by reference.

[0451] The heterodimeric bispecific antibody is based on the native IgG structure, where two binding arms recognize different antigens. By forcing heavy chain heterodimerization and combining techniques to minimize light chain (e.g., common light chain) mismatches, an IgG-derived form capable of achieving defined monovalent (and simultaneous) antigen binding is generated. Forced heavy chain heterodimerization can be achieved using, for example, the knob-in-hole structure or the strand exchange engineered domain (SEED).

[0452] Knob-in-hole structure

[0453] The knob-in-hole structure is described in US 5,731,116, US 7,476,724, and Ridgway, J. et al. (1996) Prot. Engineering 9(7):617-621 and generally involves: (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 generated by replacing a small amino acid in the parental antibody with a larger amino acid (e.g., T366Y or T366W); the "hole" or "cavity" is generated by replacing a larger residue in the parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A, and / or Y407V).

[0454] For bispecific antibodies that include an Fc domain, specific mutations introduced into the constant region of the heavy chain can be utilized to promote proper heterodimerization of the Fc portion. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the content of which is incorporated herein by reference in its entirety. These techniques include the "knob-in-hole structure" (KiH) method, which involves introducing a bulky residue into one CH3 domain of one antibody heavy chain. This bulky residue fits into a complementary "hole" in the other CH3 domain of the paired heavy chain, thereby promoting proper pairing of the heavy chains (see, for example, US7642228).

[0455] Exemplary KiH mutations include S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Table 4 provides other exemplary KiH mutations, as well as additional optional stabilizing Fc cysteine mutations.

[0456] Igawa and Tsunoda provided other Fc mutations, where they 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, either alone or in combination with the newly discovered disulfide bridge: E356K, E357K, and D399K, and at least two of the following three mutations in chain B: K370E, K409D, K439E, very efficient heterodimerization can be promoted while homodimerization is inhibited (Martens T et al. A novel one-armed anti-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, and subsequently screened for maximum heterodimerization, defining the combination of S364H, F405A (HA) on chain A and Y349T, T394F (TF) on chain B (Moore GL et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011;3:546-57; PMID:22123055).

[0457] Other exemplary Fc mutations that promote heterodimerization of multifunctional antibodies include those described in the following references, the contents of each of which are incorporated herein by reference: WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US20150353636A1, US20140199294A1, US7750128B2, US20160229915A1, US20150344570A1, US8003774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1, and US20110054151A1.

[0458] Stabilizing cysteine mutations have also been used in combination with KiH and other variants that promote Fc heterodimerization, see, e.g., US7183076. Other exemplary cysteine modifications include, for example, those disclosed in US20140348839A1, US7855275B2, and US9000130B2.

[0459] Strand exchange engineered domain (SEED)

[0460] A heterodimeric Fc platform that supports bispecific and asymmetric fusion protein design by engineering strand exchange engineered domain (SEED) C(H)3 heterodimers is known. These derivatives of human IgG and IgA C(H)3 domains form complementary human SEED C(H)3 heterodimers, which consist of alternating segments of human IgA and IgG C(H)3 sequences. When expressed in mammalian cells, the resulting pair of SEED C(H)3 domains preferentially associate to form heterodimers. The SEEDbody (Sb) fusion protein consists 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. Their respective contents are incorporated herein by reference).

[0461] Fc-containing entity (minibody)

[0462] Fc-containing entities (also known as minibodies) can be generated by fusing an scFv to the C-terminus of the constant heavy chain domain 3 (CH3-scFv) and / or to the hinge region of an antibody with a different specificity (scFv-hinge-Fc). Trivalent entities with disulfide-stabilized variable domains (without a peptide linker) fused to the C-terminus of the CH3 domain of IgG can also be prepared.

[0463] Duobody

[0464] The "Duobody" technology for generating bispecific antibodies with correct heavy chain pairing is known. The DuoBody technology involves three basic steps for generating stable bispecific human IgG1 antibodies in a post-production exchange reaction. First, two IgG1s are produced separately using standard mammalian recombinant cell lines, each IgG1 containing a single matching mutation in the third constant (CH3) domain. Subsequently, these IgG1 antibodies are purified according to standard methods for recovery and purification. After production and (post-production) purification, the two antibodies are recombined under customized laboratory conditions to yield a bispecific antibody product with very high yield (usually >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).

[0465] Electrostatic interaction

[0466] Methods for preparing multifunctional antibodies using charged amino acids with CH3 amino acid changes have been disclosed such that homodimer formation is electrostatically disfavored. EP1870459 and WO 2009089004 describe other strategies that favor heterodimer formation when co-expressing different antibody domains in host cells. In these methods, one or more residues that make up the heavy chain constant domain 3 (CH3), the CH3-CH3 interface, in two CH3 domains are replaced with charged amino acids such that homodimer formation is electrostatically disfavored and heterodimerization is electrostatically favored. Other methods for preparing multifunctional molecules using electrostatic interactions are described in the following references, the contents of each of which are incorporated herein by reference, including US20100015133, US8592562B2, US9200060B2, US20140154254A1, and US9358286A1.

[0467] Common light chain

[0468] It is necessary to avoid light chain mispairing to produce a homogeneous preparation of bispecific IgG. One of its implementation methods is by using a common light chain principle, that is, sharing a light chain but still having two binding agents with different specificities. An exemplary method of enhancing the formation of desired bispecific antibodies by a monomer mixture is to interact with each heteropolymeric variable heavy chain region of a bispecific antibody by providing a common variable light chain. The composition and method for producing a bispecific antibody with a common light chain are disclosed in, for example, US7183076B2, US20110177073A1, EP2847231A1, WO2016079081A1 and EP3055329A1, and their respective contents are incorporated herein by reference.

[0469] CrossMab

[0470] Another option for reducing light chain mispairing is CrossMab technology, which avoids nonspecific L chain mispairing by exchanging the CH1 and CL domains in the Fab of half of the bispecific antibody. Such staggered variants retain binding specificity and affinity, but make the two arms so different that L chain mispairing is prevented. CrossMab technology (as outlined in Klein et al., above) involves domain swaps between heavy and light chains to promote the formation of correct pairing. In short, in order to construct a bispecific IgG-like CrossMab antibody that can bind to two antigens by using two different light chain-heavy chain pairs, a two-step modification process is used. First, a heterodimerization method (e.g., knob-in-hole (KiH) technology) is used to engineer the dimerization interface to the C-terminus of each heavy chain to ensure that only heterodimers of two different heavy chains from one antibody (e.g., antibody A) and a second antibody (e.g., antibody B) are effectively formed. Next, the constant heavy chain 1 (CH1) domain and the constant light chain (CL) domain of one antibody (antibody A) are exchanged, while the variable heavy chain (VH) and variable light chain (VL) domains are kept the same. The exchange of CH1 and CL domains ensures that the modified antibody (antibody A) light chain can only dimerize effectively with the modified antibody (antibody A) heavy chain, while the unmodified antibody (antibody B) light chain can only dimerize effectively with the unmodified antibody (antibody B) heavy chain; therefore, only the desired bispecific CrossMab is effectively formed (see, e.g., Cain, C. SciBX 4 (28); doi: 10.1038 / scibx.2011.783, the contents of which are incorporated herein by reference).

[0471] Common heavy chain

[0472] Exemplary methods for enhancing the formation of a desired bispecific antibody from a monomer mixture are by providing a common variable heavy chain to interact with each heteromeric variable light chain region of the bispecific antibody. Compositions and methods for generating bispecific antibodies having a common heavy chain are disclosed, for example, in US20120184716, US20130317200, and US20160264685A1, the respective contents of which are incorporated herein by reference.

[0473] Amino acid modifications

[0474] Alternative compositions and methods for generating multifunctional antibodies with correct light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers having 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 interface between the light and heavy chains and establish 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 light chain rather than with each other (see, for example, W02015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

[0475] λ / κ form

[0476] Multifunctional molecules (e.g., multispecific antibody molecules) comprising a λ light chain polypeptide and a κ light chain polypeptide can be used to permit heterodimerization. Methods for generating bispecific antibody molecules comprising a λ light chain polypeptide and a κ light chain polypeptide are disclosed in PCT / US17 / 53053, filed September 22, 2017, designated publication number WO2018 / 057955, which is incorporated herein by reference in its entirety.

[0477] In some embodiments, the multifunctional molecule comprises a multispecific antibody molecule, e.g., an antibody molecule having two binding specificities, e.g., a bispecific antibody molecule. The multispecific antibody molecule comprises:

[0478] A λ light chain polypeptide 1 (LLCP1) specific for a first epitope;

[0479] A heavy chain polypeptide 1 (HCP1) specific for the first epitope;

[0480] A κ light chain polypeptide 2 (KLCP2) specific for a second epitope; and

[0481] Heavy chain polypeptide 2 (HCP2) specific for the second epitope.

[0482] As used herein, the term "lambda light chain polypeptide 1 (LLCP1)" 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 complexing with HCP1. In some embodiments, LLCP1 comprises 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 sequences therefrom sufficient to mediate specific binding to its epitope and complexing with HCP1. LLCP1, together with its HCP1, provides specificity for the first epitope (whereas KLCP2, together with its HCP2, provides specificity for the second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0483] As used herein, the term "kappa light chain polypeptide 2 (KLCP2)" 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 complexing with HCP2. In some embodiments, KLCP2 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 sequences therefrom sufficient to mediate specific binding to its epitope and complexing with HCP2. KLCP2, together with its HCP2, provides specificity for the second epitope (whereas LLCP1 and its HCP1 provide specificity for the first epitope).

[0484] As used herein, the term "heavy chain polypeptide 1 (HCP1)" refers to a polypeptide that contains sufficient heavy chain (HC) sequence (e.g., HC variable region sequence) such that when combined with its cognate LLCP1, it can mediate specific binding to its epitope and complex formation with HCP1. In some embodiments, HCP1 contains all or a fragment of the CH1 region. In some embodiments, HCP1 contains all or a fragment of the CH2 and / or CH3 regions. In some embodiments, HCP1 contains HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences from them sufficient to effect: (i) mediate specific binding to its epitope and complex formation with LLCP1, (ii) preferentially complex with LLCP1 rather than KLCP2 as described herein; and (iii) preferentially complex with another molecule of HCP2 rather than HCP1 as described herein. HCP1, together with its LLCP1, provides specificity for a first epitope (whereas KLCP2, together with its HCP2, provides specificity for a second epitope).

[0485] As used herein, the term "heavy chain polypeptide 2 (HCP2)" refers to a polypeptide that contains sufficient heavy chain (HC) sequence (e.g., HC variable region sequence) such that when combined with its cognate LLCP1, it can mediate specific binding to its epitope and complex formation with HCP1. In some embodiments, HCP2 contains all or a fragment of the CH1 region. In some embodiments, HCP2 contains all or a fragment of the CH2 and / or CH3 regions. In some embodiments, HCP1 contains HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sequences from them sufficient to effect: (i) mediate specific binding to its epitope and complex formation with KLCP2, (ii) preferentially complex with KLCP2 rather than LLCP1 as described herein; and (iii) preferentially complex with another molecule of HCP1 rather than HCP2 as described herein. HCP2, together with its KLCP2, provides specificity for a second epitope (whereas LLCP1, together with its HCP1, provides specificity for a first epitope).

[0486] In some embodiments, in the multifunctional polypeptide molecules as described herein: the affinity of LLCP1 for HCP1 is higher than its affinity for HCP2; and / or the affinity of KLCP2 for HCP2 is higher than its affinity for HCP1.

[0487] 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 (e.g., at pH 7), in saline (e.g., 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 conjugated with HCP1.

[0488] In some embodiments, in the multifunctional polypeptide molecules as described herein: the affinity of HCP1 for HCP2 is greater than its affinity for a second molecule of HCP1; and / or

[0489] the affinity of HCP2 for HCP1 is greater than its affinity for a second molecule of HCP2.

[0490] In some embodiments, the affinity of HCP1 for HCP2 is sufficiently greater than its affinity for a second molecule of HCP1 such that, under preselected conditions, e.g., in an aqueous buffer (e.g., at pH 7), in saline (e.g., 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 conjugated with HCP2.

[0491] In another aspect, a method for preparing or generating multifunctional antibody molecules is described herein. The method comprises, under conditions where (i)-(iv) associate:

[0492] (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising a first heavy chain variable region (first VH), a first CH1, one, two, three, or all of a first heavy chain constant region (e.g., first CH2, first CH3, or both));

[0493] (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising a second heavy chain variable region (second VH), a second CH1, one, two, three, or all of a second heavy chain constant region (e.g., second CH2, second CH3, or both));

[0494] (iii) providing a λ chain polypeptide that preferentially associates with the first heavy chain polypeptide (e.g., first VH) (e.g., a λ light variable region (VLλ), a λ light constant chain (CLλ), or both); and

[0495] (iv) providing a κ chain polypeptide that preferentially associates with the second heavy chain polypeptide (e.g., second VH) (e.g., a κ light variable region (VLκ), a κ light constant chain (CLκ), or both).

[0496] In some embodiments, the first heavy chain and the second heavy chain polypeptides form an Fc interface that enhances heterodimerization.

[0497] In some embodiments, (i)-(iv) (e.g., nucleic acids encoding (i)-(iv)) are introduced into a single cell, such as a single mammalian cell, such as 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, such as different mammalian cells, such as two or more CHO cells. In some embodiments, (i)-(iv) are expressed in the cells.

[0498] In some embodiments, the method further comprises purifying the antibody molecule expressed by the cell, such as using λ- and / or κ-specific purification (e.g., affinity chromatography).

[0499] In some embodiments, the method further comprises evaluating the multifunctional antibody molecule expressed by the cell. For example, the purified multifunctional antibody molecule expressed by the cell can be analyzed by techniques known in the art, including mass spectrometry. In some embodiments, the purified antibody molecule expressed by the cell is cleaved, such as digested with papain to produce Fab fragments, and evaluated using mass spectrometry.

[0500] In some embodiments, the method produces correctly paired κ / λ multispecific (e.g., bispecific) antibody molecules in high yield, such as at least 75%, 80, 90, 95, 98, 99, 99.5 or 99.9%.

[0501] In other embodiments, the multispecific (e.g., bispecific) antibody molecule comprises:

[0502] (i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising a first heavy chain variable region (first VH), a first CH1, one, two, three or all of a first heavy chain constant region (e.g., first CH2, first CH3 or both)), such as wherein HCP1 binds to a first epitope;

[0503] (ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising a second heavy chain variable region (second VH), a second CH1, one, two, three or all of a second heavy chain constant region (e.g., second CH2, second CH3 or both)), such as wherein HCP2 binds to a second epitope;

[0504] (iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light variable region (VLλ), a lambda light constant chain (VLλ), or both) that preferentially associates with a first heavy chain polypeptide (e.g., a first VH), e.g., wherein LLCP1 binds to a first epitope; and

[0505] (iv) a kappa light chain polypeptide (KLCP2) (e.g., a kappa light variable region (VLκ), a kappa light constant chain (VLκ) or both) that preferentially associates with a second heavy chain polypeptide (e.g., a second VH), e.g., wherein KLCP2 binds to a second epitope.

[0506] In some embodiments, the first heavy chain polypeptide and the second heavy chain polypeptide form an Fc interface that enhances heterodimerization.In some embodiments, the multifunctional antibody molecule has a first binding specificity comprising a hybrid VLλ-CLλ that is heterodimerized to a first heavy chain variable region (with a knob modification) connected to an Fc constant CH2-CH3 domain, and a second binding specificity comprising a hybrid VLκ-CLκ that is heterodimerized to a second heavy chain variable region (with a hole modification) connected to an Fc constant CH2-CH3 domain.

[0507] Multispecific or multifunctional antibody molecules

[0508] Exemplary structures of multispecific and multifunctional molecules defined herein are described in full. Exemplary structures are further described in the following literature: 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 which are incorporated herein by reference.

[0509] In some embodiments, a multispecific antibody molecule can comprise more than one antigen-binding site, where different sites are 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 comprises 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 divided into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0510] BsIgG is in a monovalent form for each antigen. Exemplary BsIgG forms include, but are not limited to, crossMab, DAF (dual action format), DAF (quadruple action format), DutaMab, DT-IgG, common LC of the knobs-into-holes structure, knobs-into-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kappa-lambda body, orthogonal Fab. See Spiess et al. Mol. Immunol. 67 (2015): 95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which comprises an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises heavy chains engineered for heterodimerization. For example, the "knobs-into-holes" strategy, SEED platform, common heavy chains (e.g., in kappa-lambda bodies), and the use of a heterodimeric Fc region can be used to engineer heavy chains for heterodimerization. See Spiess et al. Mol. Immunol. 67 (2015): 95-106. Strategies for avoiding heavy chain pairing of homodimers in BsIgG include knobs-into-holes, diabody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See the previous reference. BsIgG can be produced by separately expressing the component antibodies in different host cells and subsequently purifying / assembling them into BsIgG. BsIgG can also be produced by expressing the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0511] IgG appended with an additional antigen-binding portion is another form of bispecific antibody molecule. For example, a monospecific IgG can be engineered to be bispecific by appending an additional antigen-binding unit (e.g., at the N-terminus or C-terminus of the heavy or light chain). Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). See the previous reference. Examples of appended IgG forms include bispecific variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIHIgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (quadroma). See Spiess et al. Mol. Immunol. 67 (2015): 95-106. An example of IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds IL-1α and IL-1β; and ABT-122 (AbbVie), which binds TNF and IL-17A.

[0512] Bispecific antibody fragments (BsAb) are forms of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack the Fc region. In some embodiments, bispecific antibody fragments include heavy and light chain regions linked by a peptide linker that allows for efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTE, diabodies, DART, TandAb, sc diabodies, sc diabodies-CH3, diabody-CH3, triple body, minibodies, microbodies, TriBi microbodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, and intracellular antibodies. See the previous reference. For example, the BiTE form includes tandem scFv, where the component scFv binds to CD3 on T cells and a surface antigen on cancer cells.

[0513] Bispecific fusion proteins include antibody fragments linked to other proteins, e.g., to increase additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC, which comprises an anti-CD3 scFv linked to an affinity matured T cell receptor that recognizes HLA-presented peptides. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusions with albumin-binding proteins or human serum albumin can extend the serum half-life of antibody fragments. See the previous reference.

[0514] In some embodiments, chemical conjugation, e.g., of antibodies and / or antibody fragments, can be used to generate BsAb molecules. See the previous reference. Exemplary bispecific antibody conjugates include the CovX-body format, in which a low molecular weight drug is site-specifically conjugated to a single reactive lysine in each Fab arm or in the antibody or its fragment. In some embodiments, conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit VEGF or Ang2. See the previous reference.

[0515] Antibody molecules can be produced by recombinant expression of at least one or more components in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells such as CHO cells, or insect cells such as SF9 or S2 cells) and prokaryotic cells (e.g., Escherichia coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells followed by purification / assembly. Alternatively, antibody molecules can be produced by expressing the components in a single host cell. Purification of bispecific antibody molecules can be carried out by various methods such as affinity chromatography, e.g., using Protein A and continuous pH elution. In other embodiments, affinity tags can be used for purification, e.g., histidine-containing tags, myc tags or streptavidin tags.

[0516] Linker

[0517] Multispecific or multifunctional molecules as described herein may further comprise linkers, such as linkers between one or more of the following: antigen-binding domains and cytokine molecules, antigen-binding domains and immune cell adaptors, antigen-binding domains and matrix-modifying moieties, cytokine molecules and immune cell adaptors, cytokine molecules and matrix-modifying moieties, immune cell adaptors and matrix-modifying moieties, antigen-binding domains and immunoglobulin chain constant regions, cytokine molecules and immunoglobulin chain constant regions, immune cell adaptors and immunoglobulin chain constant regions, or matrix-modifying moieties and immunoglobulin chain constant regions. In some embodiments, the linker is selected from: cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, or non-helical linkers, or combinations thereof.

[0518] In some embodiments, the multispecific molecule may comprise one, two, three, or four linkers, such as peptide linkers. In some embodiments, the peptide linker comprises Gly and Ser. In some embodiments, the peptide linker is selected from GGGGS (SEQ ID NO:3307); GGGGSGGGGS (SEQ ID NO:3308); GGGGSGGGGSGGGGS (SEQ ID NO:3309); DVPSGPGGGGGSGGGGS (SEQ ID NO:3310); and GGGGSGGGGSGGGGGS (SEQ ID NO:3643). In some embodiments, the peptide linker is of the A(EAAAK)nA (SEQ ID NO:3437) family of linkers (e.g., as described in Protein Eng. (2001) 14(8):529-532). These are rigid helical linkers, where n ranges from 2-5. In some embodiments, the peptide linker is selected from AEAAAKEAAAKAAA (SEQ ID NO:3314); AEAAAKEAAAKEAAAKAAA (SEQ ID NO:3315); AEAAAKEAAAKEAAAKEAAAKAAA (SEQ ID NO:3316); and AEAAAKEAAAKEAAAKEAAAKEAAAKAAA (SEQ ID NO:3317).

[0519] Nucleic acid

[0520] In certain embodiments, an isolated nucleic acid molecule is described herein that comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to a nucleotide sequence encoding a multifunctional polypeptide molecule as described herein.

[0521] Also disclosed are nucleic acids encoding the foregoing antibody molecules, such as anti-TCRβV antibody molecules, multispecific or multifunctional molecules.

[0522] In certain embodiments, the invention features nucleic acids that comprise nucleotide sequences encoding the heavy and light chain variable regions and CDRs or hypervariable loops of the antibody molecules as described herein. For example, the invention features a first nucleic acid and a second nucleic acid that encode the heavy and light chain variable regions of an antibody molecule, respectively, selected from one or more antibody molecules as described herein. The nucleic acid may comprise a nucleotide sequence listed in the tables herein, or be substantially the same as it (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto), or a sequence that differs from the sequences shown in the tables herein by no more than 3, 6, 15, 30 or 45 nucleotides.

[0523] In certain embodiments, the nucleic acid may comprise a nucleotide sequence encoding at least one, two or three CDRs or hypervariable loops from the heavy chain variable region that has an amino acid sequence listed in the tables herein or is substantially homologous thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, and / or has one or more substitutions, such as conservative substitutions). In other embodiments, the nucleic acid may comprise a nucleotide sequence encoding at least one, two or three CDRs or hypervariable loops from the light chain variable region that has an amino acid sequence listed in the tables herein, or is substantially homologous thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, and / or has one or more substitutions, such as conservative substitutions). In yet another embodiment, the nucleic acid may comprise a nucleotide sequence encoding at least one, two, three, four, five or six CDRs or hypervariable loops from the heavy chain variable region and the light chain variable region that have amino acid sequences listed in the tables herein, or are substantially homologous thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, and / or has one or more substitutions, such as conservative substitutions).

[0524] In certain embodiments, a nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs or hypervariable loops from a heavy chain variable region having a nucleotide sequence as set forth in the tables herein, a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence capable of hybridizing under the stringent conditions described herein). In another embodiment, a nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs or hypervariable loops from a light chain variable region having a nucleotide sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence capable of hybridizing under the stringent conditions described herein). In yet another embodiment, a nucleic acid can comprise a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs or hypervariable loops from a heavy chain variable region and a light chain variable region having a nucleotide sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence capable of hybridizing under the stringent conditions described herein).

[0525] In certain embodiments, a nucleic acid can comprise a nucleotide sequence encoding a cytokine molecule, an immune cell adaptor, or a matrix modifying moiety as described herein.

[0526] In another aspect, the present application features host cells and vectors containing the nucleic acids described herein. The nucleic acids can be present in a singl...

Claims

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

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

3. A method of treating cancer in a human subject in need thereof, the method comprising administering a first dose of a multifunctional molecule to the human subject, wherein the multifunctional molecule comprises (a) a TCRβV6 binding moiety, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof, wherein the human subject is characterized as having a solid tumor, and wherein (i) if the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously received treatment for the symptomatic central nervous system (CNS) metastases, has been asymptomatic for 14 days or longer, and is not currently receiving treatment for CNS disease and does not currently have leptomeningeal disease or spinal cord compression; and (ii) if the human subject has previously received checkpoint inhibitor therapy (CPI), the CPI immune-related toxicity in the human subject has resolved to ≤ grade 1 or relative to the baseline prior to receiving the CPI treatment.

4. A method of treating cancer in a human subject in need thereof, the method comprising administering a first dose of a multifunctional molecule to the human subject, wherein the multifunctional molecule comprises (a) a TCRβV6 binding moiety, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof, wherein the human subject: (i) has no history of autoimmune disease; (ii) has not had major surgery or traumatic injury within 8 weeks prior to the first administration of the multifunctional molecule, or the subject has no unhealed wound from surgery or injury; (iii) has not received immunosuppressive drugs at a daily dose > 10 mg within 7 days prior to the first administration of the multifunctional molecule; (iv) has not previously received cytotoxic chemotherapy, small molecule inhibitors, radiotherapy, or interventional radiology procedures within 2 weeks prior to the first administration of the multifunctional molecule; (v) has not previously received monoclonal antibodies, antibody-drug conjugates, radioimmunoconjugates within 6 weeks prior to the first administration of the multifunctional molecule; (vi) has no unresolved inflammatory process within 4 weeks prior to the first administration of the multifunctional molecule; (vii) has no clinically significant pulmonary impairment; or (viii) There is no active viral, bacterial, or systemic fungal infection requiring parenteral treatment within 7 days of the first administration of the multifunctional molecule.

5. The method according to claim 1, 3, or 4, wherein the first dose is the first dose in a multi-dose.

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

7. The method according to claim 6, wherein (i) If the human subject has symptomatic central nervous system (CNS) metastases, the human subject has previously received treatment for the symptomatic CNS metastases, has been asymptomatic for 14 days or longer, and is not currently receiving treatment for CNS disease and does not currently have leptomeningeal disease or spinal cord compression; and (ii) If the human subject has previously received checkpoint inhibitor therapy (CPI), the CPI immune-related toxicity of the human subject has regressed to ≤ grade 1 or relative to the baseline before receiving the CPI treatment.

8. The method according to claim 3 or any one of claims 5-7, wherein the solid tumor is selected from high tumor mutational burden (TMB-H), microsatellite instability / DNA mismatch repair (MSI-H / dMMR), virus-related 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. The method according to claim 8, wherein the virus-related tumors include Merkel cell carcinoma, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, or vulvar cancer.

10. The method according to claim 3 or any one of claims 5-7, wherein the human subject is not simultaneously receiving treatment for CNS disease, the subject does not have leptomeningeal disease, or the subject does not have spinal cord compression.

11. The method according to claim 3 or any one of claims 5-7, wherein the CPI immune-related toxicity of the subject is ≤ grade 1 or baseline, the subject has experienced CPI-related endocrine abnormalities, or the subject has not experienced CPI-related grade 3-4 pneumonia, pericarditis / myocarditis, colitis and intestinal perforation, myositis, encephalitis, or peripheral neuropathy.

12. The method according to claim 1, 2, 3, or 5, wherein the human subject: (i) has no history of autoimmune diseases other than the following: vitiligo; psoriasis, atopic dermatitis, or other autoimmune skin conditions that do not require systemic treatment; Graves' disease, with normal thyroid function for > 4 weeks; hypothyroidism controlled by thyroid replacement; alopecia; arthritis that can be controlled without systemic therapy except for oral non-steroidal anti-inflammatory drugs, and adrenal insufficiency well controlled by replacement therapy; (ii) has not had major surgery or traumatic injury within 8 weeks before the first administration of the multifunctional molecule, or the subject has no unhealed wound from surgery or injury; (iii) Have not received immunosuppressive drug treatment at a daily dose > 10 mg within 7 days before the first administration of the multifunctional molecule; (iv) Have not previously received cytotoxic chemotherapy, small molecule inhibitors, radiotherapy, or interventional radiology procedures within 2 weeks before the first administration of the multifunctional molecule; (v) Have not previously received monoclonal antibodies, antibody-drug conjugates, radioimmunoconjugates within 6 weeks before the first administration of the multifunctional molecule; (vi) Have no unresolved inflammatory processes within 4 weeks before the first administration of the multifunctional molecule; (vii) Have no clinically significant pulmonary impairment; or (viii) Have no active viral, bacterial, or systemic fungal infections requiring parenteral treatment within 7 days before the first administration of the multifunctional molecule.

13. The method according to claim 4, 5 or 12, wherein the autoimmune disease does not include vitiligo; psoriasis, atopic dermatitis or other autoimmune skin conditions not requiring systemic treatment; Graves' disease, with normal thyroid function for > 4 weeks; hypothyroidism controlled by thyroid replacement; alopecia; arthritis controllable without systemic therapy except for oral non-steroidal anti-inflammatory drugs, and adrenal insufficiency well controlled by replacement therapy.

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

15. The method according to any one of claims 2-14, 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. The method according to claim 1 or 15, wherein the multifunctional molecule is administered at a first dose of about 0.001 mg / kg to about 1 mg / kg.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

31. The method according to claim 1 or 15, wherein 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, 0.2 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.Administered at a first dose of 88 mg / kg, 0.89 mg / kg, 0.9 mg / kg, 0.91 mg / kg, 0.92 mg / kg, 0.93 mg / kg, 0.94 mg / kg, 0.95 mg / kg, 0.96 mg / kg, 0.97 mg / kg, 0.98 mg / kg, 0.99 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, 20 mg / kg, 20.5 mg / kg, 21 mg / kg, 21.5 mg / kg, 22 mg / kg, 22.5 mg / kg, 23 mg / kg, 23.5 mg / kg, 24 mg / kg, 24.5 mg / kg, 25 mg / kg, 25.5 mg / kg, 26 mg / kg, 26.5 mg / kg, 27 mg / kg, 27.5 mg / kg, 28 mg / kg, 28.5 mg / kg, 29 mg / kg, 29.5 mg / kg, 30 mg / kg, 30.5 mg / kg, 31 mg / kg, 31.5 mg / kg, 32 mg / kg, 32.5 mg / kg, 33 mg / kg, 33.5 mg / kg, 34 mg / kg, 34.5 mg / kg, 35 mg / kg, 35.5 mg / kg, 36 mg / kg, 36.5 mg / kg, 37 mg / kg, 37.5 mg / kg, 38 mg / kg, 38.5 mg / kg, 39 mg / kg, 39.5 mg / kg, 40 mg / kg, 40.5 mg / kg, 41 mg / kg, 41.5 mg / kg, 42 mg / kg, 42.5 mg / kg, 43 mg / kg, 43.5 mg / kg, 44 mg / kg, 44.5 mg / kg, 45 mg / kg, 45.5 mg / kg, 46 mg / kg, 46.5 mg / kg, 47 mg / kg, 47.5 mg / kg, 48 mg / kg, 48.5 mg / kg, 49 mg / kg, 49.5 mg / kg or 50 mg / kg.

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

33. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are lower than the previous dose immediately preceding the subsequent dose after indication of intolerance to the administration of the previous dose.

34. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are the same as the previous dose immediately preceding the subsequent dose after indication of tolerance to the administration of the previous dose.

35. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are higher than the previous dose immediately preceding the subsequent dose after indication of tolerance to the administration of the previous dose.

36. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are the same as the previous dose immediately preceding the subsequent dose after indication of effectiveness of the administration of the previous dose.

37. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are lower than the previous dose immediately preceding the subsequent dose after indication of effectiveness of the administration of the previous dose.

38. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are higher than the previous dose immediately preceding the subsequent dose after indication of ineffectiveness of the administration of the previous dose.

39. The method according to claim 2 or 32, wherein subsequent doses in the multiple doses are 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 the administration of the previous dose immediately preceding the subsequent dose.

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

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

42. The method according to claim 2 or 32, wherein the dosing frequency of the multiple doses is maintained or reduced after indicating the effectiveness of the previous dose immediately preceding the subsequent dose.

43. The method according to claim 2 or 32, wherein the dosing frequency of the administration is increased after indicating the ineffectiveness of a dose in the multiple doses.

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

45. The method according to claim 44, wherein the method comprises 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. The method according to any one of claims 1-43, wherein the method comprises administering the multifunctional molecule to the human subject once every two weeks.

47. The method according to claim 46, wherein the method comprises 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. The method according to any one of claims 1-43, wherein the method comprises administering the multifunctional molecule to the human subject once every three weeks.

49. The method according to claim 48, wherein the method comprises 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. The method according to any one of claims 1-43, wherein the method comprises administering the multifunctional molecule to the human subject once every two weeks for 28 days, wherein the multifunctional molecule is administered to the human subject on day 1 and day 15.

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

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

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

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

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

56. The method according to any one of claims 1-50, 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. The method according to any one of claims 1-50, 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. The method according to any one of claims 1-57, wherein the method further comprises administering at least one additional therapeutic agent or therapy.

59. The method according to claim 58, wherein the at least one additional therapeutic agent or therapy is administered concurrently with a dose of the multifunctional molecule.

60. The method according to claim 58, wherein the at least one additional therapeutic agent or therapy is administered prior to administering a dose of the multifunctional molecule.

61. The method according to claim 58, wherein the at least one additional therapeutic agent or therapy is administered after administering a dose of the multifunctional molecule.

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

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

64. The method according to claim 62, wherein the pharmaceutical composition comprises a pharmaceutically acceptable diluent that is a saline solution.

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

66. The method according to 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. The method according to 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. The method according to 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. The method according to 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 method according to claim 66, wherein the multifunctional molecule is present in the pharmaceutical composition at a concentration of 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, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.05 mg / mL, 1.1 mg / mL, 1.15 mg / mL, 1.2 mg / mL, 1.25 mg / mL, 1.3 mg / mL, 1.35 mg / mL, 1.4 mg / mL, 1.45 mg / mL, 1.5 mg / mL, 1.55 mg / mL, 1.6 mg / mL, 1.65 mg / mL, 1.7 mg / mL, 1.75 mg / mL, 1.8 mg / mL, 1.85 mg / mL, 1.9 mg / mL, 1.95 mg / mL, 2 mg / mL, 2.05 mg / mL, 2.1 mg / mL, 2.15 mg / mL, 2.2 mg / mL, 2.25 mg / mL, 2.3 mg / mL, 2.35 mg / mL, 2.4 mg / mL, 2.45 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 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. The method according to any one of claims 62 - 70, wherein the pharmaceutical composition comprises from about 0.5 mL to about 500 mL of a diluent.

72. A dosage of a pharmaceutical composition comprising a multifunctional molecule, wherein the multifunctional molecule comprises (a) a TCRβV6 binding moiety, and (b) Interleukin-2 (IL-2) or a functional fragment or variant thereof, wherein the dose is about 0.001 mg / kg to about 10 mg / kg of the multifunctional molecule.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

88. The dosage according to claim 72, wherein the dosage is about 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.The multifunctional molecule at 89 mg / kg, 0.9 mg / kg, 0.91 mg / kg, 0.92 mg / kg, 0.93 mg / kg, 0.94 mg / kg, 0.95 mg / kg, 0.96 mg / kg, 0.97 mg / kg, 0.98 mg / kg, 0.99 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, 20 mg / kg, 20.5 mg / kg, 21 mg / kg, 21.5 mg / kg, 22 mg / kg, 22.5 mg / kg, 23 mg / kg, 23.5 mg / kg, 24 mg / kg, 24.5 mg / kg, 25 mg / kg, 25.5 mg / kg, 26 mg / kg, 26.5 mg / kg, 27 mg / kg, 27.5 mg / kg, 28 mg / kg, 28.5 mg / kg, 29 mg / kg, 29.5 mg / kg, 30 mg / kg, 30.5 mg / kg, 31 mg / kg, 31.5 mg / kg, 32 mg / kg, 32.5 mg / kg, 33 mg / kg, 33.5 mg / kg, 34 mg / kg, 34.5 mg / kg, 35 mg / kg, 35.5 mg / kg, 36 mg / kg, 36.5 mg / kg, 37 mg / kg, 37.5 mg / kg, 38 mg / kg, 38.5 mg / kg, 39 mg / kg, 39.5 mg / kg, 40 mg / kg, 40.5 mg / kg, 41 mg / kg, 41.5 mg / kg, 42 mg / kg, 42.5 mg / kg, 43 mg / kg, 43.5 mg / kg, 44 mg / kg, 44.5 mg / kg, 45 mg / kg, 45.5 mg / kg, 46 mg / kg, 46.5 mg / kg, 47 mg / kg, 47.5 mg / kg, 48 mg / kg, 48.5 mg / kg, 49 mg / kg, 49.5 mg / kg or 50 mg / kg.

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

90. The pharmaceutical composition according to claim 89, wherein the pharmaceutically acceptable diluent is a 0.9% saline solution.

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

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

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

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

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

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

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

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

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

102. The pharmaceutical composition according to claim 99 or 100, wherein the pharmaceutical composition comprises about 0.5 mg to about 100 mg of the multifunctional molecule.

103. The pharmaceutical composition according to claim 99 or 100, wherein the pharmaceutical composition comprises from about 1 mg to about 200 mg of the multifunctional molecule.

104. The pharmaceutical composition according to claim 99 or 100, wherein the pharmaceutical composition comprises from 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 moiety, and (b) interleukin-2 (IL-2) or a functional fragment or functional variant thereof, wherein the pharmaceutically acceptable excipient comprises one or more of an L-histidine / L-histidine monohydrochloride buffer, sucrose, or polysorbate.

106. The pharmaceutical composition according to claim 105, wherein the pharmaceutical composition comprises from about 0.1 mg to about 500 mg of the multifunctional molecule.

107. The pharmaceutical composition according to claim 105, wherein the pharmaceutical composition comprises from about 0.5 mg to about 200 mg of the multifunctional molecule.

108. The pharmaceutical composition according to claim 105, wherein the pharmaceutical composition comprises from about 0.5 mg to about 100 mg of the multifunctional molecule.

109. The pharmaceutical composition according to claim 105, wherein the pharmaceutical composition comprises from about 1 mg to about 200 mg of the multifunctional molecule.

110. The pharmaceutical composition according to claim 105, wherein the pharmaceutical composition comprises from about 1 mg to about 100 mg of the multifunctional molecule.

111. The pharmaceutical composition according to any one of claims 105-110, wherein the pharmaceutical composition comprises from 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 of an L-histidine / L-histidine monohydrochloride buffer.

112. The pharmaceutical composition according to claim 105 or 111, wherein the pharmaceutical composition comprises from about 1% (w / v) to about 20% (w / v), about 2% (w / v) to about 15% (w / v), 5% (w / v) to about 12% (w / v), about 6% (w / v) to about 10% (w / v), about 8% (w / v) of sucrose.

113. The pharmaceutical composition according to any one of claims 105-112, wherein the pharmaceutical composition comprises from about 0.001% (w / v) to about 0.1% (w / v), about 0.002% (w / v) to about 0.08% (w / v), 0.005% (w / v) to about 0.06% (w / v), about 0.008% (w / v) to about 0.04% (w / v), about 0.01% (w / v) to about 0.03% (w / v), about 0.02% (w / v) of polysorbate-80.

114. The pharmaceutical composition according to any one of claims 105-113, wherein 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.

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

116. The method according to any one of claims 1-71, the dosage according to any one of claims 72-88, or the pharmaceutical composition according to any one of claims 89-115, wherein the multifunctional molecule comprises a first polypeptide, a second polypeptide, and a third polypeptide; wherein the first polypeptide, the second polypeptide, and the third polypeptide are discontinuous, wherein (i) the first polypeptide comprises a first part of a dimerization module, the first part of the dimerization module being linked to a first part of the TCRβV6 binding part, the first part of the TCRβV6 binding part comprising the VH of the TCRβV6 binding part; (ii) the second polypeptide comprises a second part of the dimerization module, wherein the IL-2 or a functional fragment or functional variant thereof is covalently linked to the second polypeptide; and (iii) the third polypeptide comprises a second part of the TCRβV6 binding part, the second part of the TCRβV6 binding part comprising the VL of the TCRβV6 binding part.

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

119. The method, dosage or pharmaceutical composition according to claim 118, wherein the first polypeptide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NO: 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 NO: 4020, 4022, 4024, 4026 and 4028.

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