Novel bispecific anti-CD3 / CD20 polypeptide complex formulations

Abstract

The present invention provides a novel bispecific anti-CD3 / CD20 polypeptide complex formulation comprising a first antigen-binding portion and a second antigen-binding portion, in particular, comprising a liquid formulation and a lyophilized formulation.

Description

[0001] This application is a divisional application of the invention application with a filing date of July 26, 2021, Chinese application number 202180041422.1, and invention name “Novel bispecific anti-CD3 / CD20 polypeptide complex formulation”.

[0002] Citation of Related Applications

[0003] This disclosure claims priority to Chinese Patent Application No. 202010733597.6 filed on July 27, 2020, the contents of which are incorporated by reference into this disclosure in their entirety and for all purposes. Technical Field

[0004] The present disclosure belongs to the field of pharmaceutical preparations, and in particular, relates to a bispecific anti-CD3 / CD20 polypeptide complex formulation and use thereof. Background Art

[0005] The statements of the background art merely provide information related to the present disclosure and do not necessarily constitute prior art.

[0006] With the continuous development of biological drugs, therapeutic antibodies have become the main choice of drugs for patients with cancer, autoimmune diseases, inflammation and other diseases. However, monoclonal antibodies target a single target, while the occurrence of diseases such as cancer is multifactorial, with multiple signaling pathways involved in the occurrence of the disease. Single-target immunotherapy does not seem to be enough to destroy cancer cells.

[0007] CD20 is an activated glycosylated phosphoprotein expressed on the surface of B lymphocytes. As early as 1997, the FDA approved the use of rituximab (Rituximab, a chimeric anti-CD20 monoclonal antibody (hereinafter also referred to as "mAb")) to treat lymphoproliferative disorders. With the development of research on the protein structure and molecular function of CD20, a new generation of CD20-targeted therapeutic antibodies ofatumumab (Ofatumumab, a fully human anti-CD20 therapeutic antibody) has been produced. Compared with rituximab, it targets different CD20 epitopes closer to the cell surface, resulting in a slower dissociation rate and more stable binding than rituximab (Laurenti L, Innocenti I, Autore F, et al. New developments in the management of chronic lymphocytic leukemia: role of ofatumumab. Onco Targets Ther. 2016 Jan 20; 9: 421-9). However, despite anti-CD20 mAb treatment, nearly all patients with follicular lymphoma and CLL, and approximately half of patients with aggressive B-cell lymphomas (e.g., diffuse large B-cell lymphoma) will still experience disease relapse or recurrence (Lim SH, Beers SA, French RR, et al. Anti-CD20 monoclonal antibodies: historical and future perspectives. Haematologica. 2010 Jan;95(1):135-43).

[0008] The T cell CD3 receptor is a protein complex composed of four different chains (CD3γ chain, CD3δ chain and two CD3ε chains). These four chains associate with a molecule called the T cell receptor (TCR) and an intracellular ζ chain to generate an activation signal in T lymphocytes. TCR, ζ chain and CD3 molecules form the TCR complex, in which TCR is used as a subunit to recognize and bind antigens, and CD3 is responsible for transmitting antigen stimulation to the signal transduction pathway as a subunit, and ultimately regulating T cell activity. CD3 protein is present in almost all T cells. The CD3-TCR complex regulates T cell functions in inherent and adaptive immune responses, as well as cellular and humoral immune functions, including the elimination of pathogens and the control of tumor growth through cytotoxic effects. Mouse monoclonal antibodies targeting human CD3, such as OKT3 (Kung et al., Science, 206: 347-9 (1979)), are the first generation of CD3 antibodies developed for treatment. Although OKT3 has strong immunosuppressive effects, its clinical application is hampered by severe side effects associated with its immunogenicity and mitogenic potential (Chatenoud, Nature Reviews, 3: 123-132 (2003)). OKT3 induces anti-antibody responses, promoting its own rapid clearance and neutralization (Chatenoud et al., Eur. J. Immunol., 137: 830-8 (1982)). In addition, OKT3 induces massive release of cytokines in vivo (Hirsch et al., J. Immunol, 142: 737-43 (1989)).

[0009] Bispecific antibodies targeting CD3 and CD20 can bind to T cells and B cells at the same time. Once the bispecific antibody binds to CD3-positive T cells and CD20-positive B cells at the same time, it will promote the formation of cytolytic synapses, and cytotoxic T cells will release perforins and granzymes to induce apoptosis of B cells. However, bispecific antibody drugs have a large molecular weight and a complex structure. They are easily degraded, aggregated, or undergo undesirable chemical modifications, etc., and become unstable, especially under high temperature conditions or during shear stress (for example, during transportation). In order to make bispecific antibodies suitable for production and administration, and to maintain biological activity and stability during storage and subsequent use, it is particularly important to develop stable bispecific antibody drug preparations. Summary of the invention

[0010] According to the present disclosure, the bispecific anti-CD3 / CD20 polypeptide complex can be formulated in the form of a liquid or a lyophilized powder. The formulation of the present disclosure can achieve a stable effect, wherein the bispecific anti-CD3 / CD20 polypeptide complex substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Specifically, the formulation of the present disclosure has a low level of bispecific anti-CD3 / CD20 polypeptide complex aggregation, and has fewer MFI particles and insoluble particles, and has good biological activity, and is suitable for administration in subjects.

[0011] In one aspect, the present disclosure provides a liquid formulation comprising a bispecific anti-CD3 / CD20 polypeptide complex and a buffer, wherein the buffer comprises one or more combinations of a histidine buffer, a succinate buffer, and a citrate buffer solution.

[0012] In some embodiments, the histidine salt buffer is a histidine-histidine hydrochloride buffer, the succinate buffer is a succinic acid-sodium succinate buffer, and the citrate buffer is a citric acid-sodium citrate buffer.

[0013] In some embodiments, the buffer in the aforementioned liquid formulation is a combination of one or more of histidine-histidine hydrochloride, succinic acid-sodium succinate, citric acid-sodium citrate buffer. In some embodiments, the buffer in the aforementioned liquid formulation is citric acid-sodium citrate buffer.

[0014] In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 0.1 mg / ml to about 50 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 0.5 mg / ml to about 20 mg / ml, for example, about 0.5 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml. ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 1 mg / ml to about 20 mg / ml, about 2 mg / ml to about 10 mg / ml, about 2 mg / ml to about 5 mg / ml, or about 2 mg / ml to about 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 2 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 5 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is about 10 mg / ml.

[0015] In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 0.1 mg / ml to 50 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 0.5 mg / ml to 20 mg / ml, for example, 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 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, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 1 mg / ml to 20 mg / ml, 2 mg / ml to 10 mg / ml, 2 mg / ml to 5 mg / ml, or 2 mg / ml to 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 2 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 5 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned liquid formulation is 10 mg / ml.

[0016] In some embodiments, the pH of the buffer in the aforementioned liquid formulation is about 5.0 to about 7.5. In some embodiments, the pH of the buffer in the aforementioned liquid formulation is about 5.0 to about 6.5, for example, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5. In some embodiments, the pH of the buffer in the aforementioned liquid formulation is about 6.0 to about 6.5. In some embodiments, the pH of the buffer in the aforementioned liquid formulation is about 6. In some embodiments, the pH of the buffer in the aforementioned liquid formulation is about 6.2. In some embodiments, the pH of the buffer in the aforementioned liquid formulation is about 6.5.

[0017] In some embodiments, the pH of the buffer described in the aforementioned liquid formulation is 5.0 to 7.5. In some embodiments, the pH of the buffer described in the aforementioned liquid formulation is 5.0 to 6.5, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5. In some embodiments, the pH of the buffer described in the aforementioned liquid formulation is 6.0 to 6.5. In some embodiments, the pH of the buffer described in the aforementioned liquid formulation is 6. In some embodiments, the pH of the buffer described in the aforementioned liquid formulation is 6.2. In some embodiments, the pH of the buffer described in the aforementioned liquid formulation is 6.5.

[0018] In some embodiments, the concentration of the buffer in the aforementioned liquid formulation is about 1mM to about 30mM. In some embodiments, the concentration of the buffer in the aforementioned liquid formulation is about 5mM to about 20mM, for example, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM. In some embodiments, the concentration of the buffer in the aforementioned liquid formulation is about 10mM.

[0019] In some embodiments, the concentration of the buffer in the aforementioned liquid formulation is 1mM to 30mM. In some embodiments, the concentration of the buffer in the aforementioned liquid formulation is 5mM to 20mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM. In some embodiments, the concentration of the buffer in the aforementioned liquid formulation is 10mM.

[0020] In some embodiments, the pH of the aforementioned liquid formulation is about 5.0 to about 7.5. In some embodiments, the pH of the aforementioned liquid formulation is about 5.0 to about 6.5, for example, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5. In some embodiments, the pH of the aforementioned liquid formulation is about 6.0 to about 6.5. In some embodiments, the pH of the aforementioned liquid formulation is about 6. In some embodiments, the pH of the aforementioned liquid formulation is about 6.2. In some embodiments, the pH of the aforementioned liquid formulation is about 6.5.

[0021] In some embodiments, the pH of the aforementioned liquid formulation is 5.0 to 7.5. In some embodiments, the pH of the aforementioned liquid formulation is 5.0 to 6.5, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5. In some embodiments, the pH of the aforementioned liquid formulation is 6.0 to 6.5. In some embodiments, the pH of the aforementioned liquid formulation is 6. In some embodiments, the pH of the aforementioned liquid formulation is 6.2. In some embodiments, the pH of the aforementioned liquid formulation is 6.5.

[0022] In some embodiments, the aforementioned liquid formulation also includes an osmotic pressure regulator and / or a stabilizer. In some embodiments, the osmotic pressure regulator and / or a stabilizer include sugar. "Sugar" used in the present disclosure can be monosaccharide, disaccharide, trisaccharide, polysaccharide, sugar alcohol, reducing sugar and non-reducing sugar, such as glucose, sucrose, trehalose, lactose, fructose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose etc. In some embodiments, the osmotic pressure regulator and / or a stabilizer also include one or more of sodium chloride, arginine-hydrochloride, glycine or proline. In some embodiments, the osmotic pressure regulator and / or a stabilizer in the aforementioned liquid formulation is a disaccharide, such as trehalose or sucrose. In some embodiments, the aforementioned liquid formulation contains trehalose. In some embodiments, the aforementioned liquid formulation contains trehalose, and one or more of sodium chloride, arginine-hydrochloride, glycine or proline. In some embodiments, the aforementioned liquid formulation contains sucrose, and one or more of sodium chloride, arginine-hydrochloride, glycine or proline. In some embodiments, the aforementioned liquid formulation contains trehalose and sodium chloride. In some embodiments, the aforementioned liquid formulation contains trehalose and arginine-hydrochloride. In some embodiments, the aforementioned liquid formulation contains sucrose and sodium chloride. In some embodiments, the aforementioned liquid formulation contains sucrose and sodium chloride. In some embodiments, the aforementioned liquid formulation contains sucrose and arginine-hydrochloride. In some embodiments, the aforementioned liquid formulation contains sucrose and arginine-hydrochloride. In the formulation of the present application, sugar includes sugar and / or its hydrate, such as trehalose including trehalose and / or its hydrate, such as dihydrated trehalose.

[0023] In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 150mM to about 320mM. In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 180mM to about 265mM, for example, about 180mM, about 185mM, about 198mM, about 211mM, about 214mM, about 217mM, about 219mM, about 222mM, about 225mM, about 227mM, about 230mM, about 233mM, about 235mM, about 238mM, about 241mM, about 243mM, about 246mM, about 248mM, about 251mM, 254mM, about 256mM, about 259mM, about 262mM, about 264mM. In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 230mM to about 240mM, or about 230mM to about 235mM. In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 233mM. In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 234mM. In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 180mM to about 265mM, for example, about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39mM, about 222.03mM, about 224.67mM, about 227.31mM, about In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 232.6mM. In some embodiments, the concentration of sugar in the aforementioned liquid formulation is about 233.7mM.

[0024] In some embodiments, the concentration of trehalose in the aforementioned liquid formulation is about 150mM to about 320mM. In some embodiments, the concentration of trehalose in the aforementioned liquid formulation is about 180mM to about 265mM, for example, about 180mM, about 185mM, about 198mM, about 211mM, about 214mM, about 217mM, about 219mM, about 222mM, about 225mM, about 227mM, about 230mM, about 233mM, about 235mM, about 238mM, about 241mM, about 243mM, about 246mM, about 248mM, about 251mM, 254mM, about 256mM, about 259mM, about 262mM, about 264mM. In some embodiments, the concentration of trehalose in the aforementioned liquid formulation is about 230mM to about 240mM, or about 230mM to about 235mM. In some embodiments, the concentration of trehalose in the aforementioned liquid formulation is about 233mM. In some embodiments, the concentration of trehalose in the aforementioned liquid formulation is about 180mM to about 265mM, for example, about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39mM, about 222.03mM, about 224.67mM, about 227.31mM, About 229.96mM, about 232.6mM, about 235.24mM, about 237.89mM, about 240.53mM, about 243.17mM, about 245.82mM, about 248.46mM, about 251.1mM, 253.75mM, about 256.39mM, about 259.03mM, about 261.68mM, about 264.32mM. In some embodiments, the concentration of trehalose in the aforementioned liquid formulation is about 232.6mM.

[0025] In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned liquid formulation is about 10mM to about 300mM. In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned liquid formulation is about 20mM to about 200mM, for example, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned liquid formulation is about 50mM to about 150mM, about 100mM to about 110mM, or about 100mM to about 105mM. In some embodiments, the concentration of sodium chloride in the aforementioned liquid formulation is about 102.7mM. In some embodiments, the concentration of sodium chloride in the aforementioned liquid formulation is about 103mM. In some embodiments, the concentration of sodium chloride in the aforementioned liquid formulation is about 6 mg / ml.In some embodiments, the concentration of arginine-HCl in the aforementioned liquid formulation is about 100 mM.

[0026] In some embodiments, the concentration of sodium chloride, arginine-HCl, glycine, or proline in the aforementioned liquid formulation is 10 mM to 300 mM. In some embodiments, the concentration of sodium chloride, arginine-HCl, glycine or proline in the aforementioned liquid formulation is 20mM to 200mM, for example, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM. In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned liquid formulation is 50mM to 150mM, 100mM to 110mM, or 100mM to 105mM. In some embodiments, the concentration of sodium chloride in the aforementioned liquid formulation is about 102.7mM. In some embodiments, the concentration of sodium chloride in the aforementioned liquid formulation is about 103mM. In some embodiments, the concentration of sodium chloride in the aforementioned liquid formulation is 6mg / ml. In some embodiments, the concentration of arginine-hydrochloride in the aforementioned liquid formulation is 100mM.

[0027] In some embodiments, the aforementioned liquid formulation further comprises a surfactant, which can be selected from polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-sweet Betaine, cetyl-betaine, lauroyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20. In some embodiments, the aforementioned liquid formulation comprises polysorbate 80 (also known as Tween 80).

[0028] In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is about 0.001% to about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is about 0.005% to about 0.08% (w / v), for example, about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.0 15% (w / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is about 0.01% to about 0.04% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is about 0.02% (w / v).

[0029] In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is 0.001% to 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is 0.005% to 0.08% (w / v), for example, 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), 0.01% (w / v), 0.015% (w / v), 0.02% (w / v), 0.025% (w / v), 0.03% (w / v), 0.035% (w / v), 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is 0.01% to 0.04% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned liquid formulation is 0.02% (w / v).

[0030] On the one hand, the present disclosure provides a liquid formulation comprising a bispecific anti-CD3 / CD20 polypeptide complex, and one or more of a buffer, an osmotic pressure regulator and / or a stabilizer, and / or a surfactant. In some embodiments, the liquid formulation of the present disclosure comprises a bispecific anti-CD3 / CD20 polypeptide complex, and a buffer, an osmotic pressure regulator and / or a stabilizer, and a surfactant.

[0031] In some embodiments, the aforementioned liquid formulation comprises:

[0032] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0033] (b) about 1 mM to about 30 mM citrate buffer,

[0034] (c) about 150 mM to about 320 mM of a sugar, and optionally, one or more of about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and

[0035] (d) from about 0.001% to about 0.1% (w / v) of a surfactant,

[0036] The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0037] In some embodiments, the aforementioned liquid formulation comprises:

[0038] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0039] (b) about 1 mM to about 30 mM citrate buffer,

[0040] (c) about 150 mM to about 320 mM trehalose, and optionally, one or more of about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and

[0041] (d) about 0.001% to about 0.1% (w / v) polysorbate 80,

[0042] The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0043] In some embodiments, the aforementioned liquid formulation comprises:

[0044] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0045] (b) about 5 mM to about 20 mM citrate buffer,

[0046] (c) about 180 mM to about 265 mM trehalose, and optionally, one or more of about 20 mM to about 200 mM sodium chloride, about 20 mM to about 200 mM arginine-hydrochloride, about 20 mM to about 200 mM glycine, or about 20 mM to about 200 mM proline, and

[0047] (d) about 0.005% to about 0.08% (w / v) polysorbate 80,

[0048] wherein the pH of the liquid formulation is from about 6.0 to about 6.5.

[0049] In some embodiments, the aforementioned liquid formulation comprises:

[0050] (a) about 0.5 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex (e.g., about 0.5 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml),

[0051] (b) about 5 mM to about 20 mM citric acid-sodium citrate buffer (e.g., about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM),

[0052] (c) about 180 mM to about 265 mM trehalose (e.g., about 180 mM, about 185 mM, about 198 mM, about 211 mM, about 214 mM, about 217 mM, about 219 mM, about 222 mM, about 225 mM, about 227 mM, about 230 mM, about 233 mM, about 235 mM, about 238 mM, about 241 mM , about 243mM, about 246mM, about 248mM, about 251mM, 254mM, about 256mM, about 259mM, about 262mM, about 264mM; or about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39m M, about 222.03mM, about 224.67mM, about 227.31mM, about 229.96mM, about 232.6mM, about 235.24mM, about 237.89mM, about 240.53mM, about 243.17mM, about 245.82mM, about 248.46mM, about 251.1mM, 253.75mM, about 256.39mM, about 259.03mM, about 261.68mM, about 264.32mM), and optionally, may further comprise one or more of about 20mM to about 200mM sodium chloride, about 20mM to about 200mM arginine-hydrochloride, about 20mM to about 200mM glycine, or about 20mM to about 200mM proline, and

[0053] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 (e.g., about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.015% (w / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v)),

[0054] wherein the pH of the liquid formulation is about 6.0 to about 6.5 (eg, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5).

[0055] In some embodiments, the aforementioned liquid formulation comprises:

[0056] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0057] (b) a buffer of about 1 mM to about 30 mM (e.g., one or more of histidine-histidine hydrochloride buffer, succinic acid-sodium succinate buffer, and citric acid-sodium citrate buffer),

[0058] (c) about 150 mM to about 320 mM of a sugar, and about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and

[0059] (d) from about 0.001% to about 0.1% (w / v) of a surfactant,

[0060] The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0061] In some embodiments, the aforementioned liquid formulation comprises:

[0062] (a) about 0.5 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex (e.g., about 0.5 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml),

[0063] (b) about 5 mM to about 20 mM histidine-histidine hydrochloride or citrate-sodium citrate buffer (e.g., about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM),

[0064] (c) about 180 mM to about 265 mM trehalose or sucrose (e.g., about 180 mM, about 185 mM, about 198 mM, about 211 mM, about 214 mM, about 217 mM, about 219 mM, about 222 mM, about 225 mM, about 227 mM, about 230 mM, about 233 mM, about 235 mM, about 238 mM, about 241 mM, about 243 mM, about 246 mM, about 248 mM, about 251 mM, 254 mM, about 256 mM) or about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39mM, about 222.03mM, about 224.67mM, about 227.31mM, about 229.96mM, about 232.6mM, about 235.24mM, about 237.89mM, about 240.53mM, about about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 61 mM, about 62 mM, about 64 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 100 mM M, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 130mM, about 135mM, about 140mM, about 145mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM), and

[0065] (d) about 0.005% to about 0.08% (w / v) of polysorbate 80 or polysorbate 20 (e.g., about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.015% (w / v) / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v)),

[0066] The pH of the liquid formulation is about 6.0 to about 6.5 (eg, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5).

[0067] In some embodiments, the aforementioned liquid formulation comprises:

[0068] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0069] (b) about 1 mM to about 30 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer,

[0070] (c) about 150 mM to about 320 mM trehalose or sucrose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0071] (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20,

[0072] The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0073] In some embodiments, the aforementioned liquid formulation comprises:

[0074] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0075] (b) about 5 mM to about 20 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer,

[0076] (c) about 180 mM to about 265 mM trehalose or sucrose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0077] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20,

[0078] wherein the pH of the liquid formulation is from about 6.0 to about 6.5.

[0079] In some embodiments, the aforementioned liquid formulation comprises:

[0080] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0081] (b) about 1 mM to about 30 mM citric acid-sodium citrate buffer,

[0082] (c) about 150 mM to about 320 mM trehalose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0083] (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20,

[0084] The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0085] In some embodiments, the aforementioned liquid formulation comprises:

[0086] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0087] (b) about 5 mM to about 20 mM citric acid-sodium citrate buffer,

[0088] (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0089] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20,

[0090] wherein the pH of the liquid formulation is from about 6.0 to about 6.5.

[0091] In some embodiments, the aforementioned liquid formulation comprises:

[0092] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0093] (b) about 1 mM to about 30 mM citrate buffer,

[0094] (c) about 150 mM to about 320 mM trehalose, and about 10 mM to about 300 mM sodium chloride, and (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20, wherein the pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0095] In some embodiments, the aforementioned liquid formulation comprises:

[0096] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0097] (b) about 5 mM to about 20 mM citrate buffer,

[0098] (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride, and (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20, wherein the pH of the liquid formulation is about 6.0 to about 6.5.

[0099] In some embodiments, the aforementioned liquid formulation comprises:

[0100] (a) about 1 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0101] (b) about 5 mM to about 20 mM citrate buffer,

[0102] (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride, and (d) about 0.01% to about 0.04% (w / v) polysorbate 80 or polysorbate 20, wherein the pH of the liquid formulation is about 6.0 to about 6.5.

[0103] In some embodiments, the aforementioned liquid formulation comprises:

[0104] (a) about 2 mg / ml to about 3 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0105] (b) about 10 mM citric acid-sodium citrate buffer,

[0106] (c) about 233 mM trehalose, and

[0107] (d) about 0.02% (w / v) polysorbate 80,

[0108] wherein the pH of the liquid formulation is about 6.2.

[0109] In some embodiments, the aforementioned liquid formulation comprises:

[0110] (a) about 2 mg / ml to about 3 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0111] (b) about 10 mM citric acid-sodium citrate buffer,

[0112] (c) about 232.6 mM trehalose, and

[0113] (d) about 0.02% (w / v) polysorbate 80,

[0114] The pH of the liquid formulation is about 6.2.

[0115] In some embodiments, the aforementioned liquid formulation comprises:

[0116] (a) about 5 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0117] (b) about 10 mM citrate buffer,

[0118] (c) about 233 mM trehalose, and about 103 mM sodium chloride, and

[0119] (d) about 0.02% (w / v) polysorbate 80,

[0120] The pH of the liquid formulation is about 6.

[0121] In some embodiments, the aforementioned liquid formulation comprises:

[0122] (a) about 5 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0123] (b) about 10 mM citrate buffer,

[0124] (c) about 232.6 mM trehalose, and about 102.7 mM sodium chloride, and

[0125] (d) about 0.02% (w / v) polysorbate 80,

[0126] The pH of the liquid formulation is about 6.

[0127] In some embodiments, the aforementioned liquid formulation comprises:

[0128] (a) about 5 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0129] (b) about 10 mM citrate buffer,

[0130] (c) about 88 mg / ml of trehalose dihydrate, and about 6 mg / ml of sodium chloride, and

[0131] (d) about 0.02% (w / v) polysorbate 80,

[0132] The pH of the liquid formulation is about 6.

[0133] In some embodiments, the aforementioned liquid formulation comprises:

[0134] (a) about 5 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0135] (b) about 10 mM citric acid-sodium citrate buffer,

[0136] (c) about 88 mg / ml of trehalose dihydrate, and about 6 mg / ml of sodium chloride, and

[0137] (d) about 0.02% (w / v) polysorbate 80,

[0138] The pH of the liquid formulation is about 6.

[0139] The present disclosure also provides a method for preparing the aforementioned liquid formulation, which includes the step of contacting the aforementioned bispecific anti-CD3 / CD20 polypeptide complex with a buffer, for example, replacing the bispecific anti-CD3 / CD20 polypeptide complex into a buffer, the buffer is preferably a citrate buffer, more preferably a citric acid-sodium citrate buffer, the concentration of the buffer is about 5mM to about 20mM, for example, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM. In some embodiments, the concentration of the liquid is about 10mM. The pH of the buffer is about 5.0 to about 6.5, for example, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5. In some embodiments, the pH of the buffer is about 6. In some embodiments, the pH of the buffer is about 6.2. In some embodiments, the pH of the buffer is about 6.5.

[0140] The present disclosure also provides a method for preparing the aforementioned liquid formulation, which comprises contacting the aforementioned bispecific anti-CD3 / CD20 polypeptide complex with a buffer, and further comprises adding an osmotic pressure regulator and / or a stabilizer and a surfactant, such as trehalose and polysorbate 80, to the resulting solution, and optionally, one or more of sodium chloride, arginine-hydrochloride, glycine or proline can also be added, regardless of the order, wherein the concentration of the buffer is preferably about 5mM to about 20mM, such as about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM. In some embodiments, the concentration of the buffer is about 10mM. The pH of the buffer is about 5.0 to about 6.5, for example, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5. In some embodiments, the pH of the buffer is about 6. In some embodiments, the pH of the buffer is about 6.2. In some embodiments, the pH of the buffer is about 6.5.

[0141] The present disclosure also provides a method for preparing a lyophilized preparation containing a bispecific anti-CD3 / CD20 polypeptide complex, which includes the step of freeze-drying the aforementioned liquid formulation. In some embodiments, the freeze-drying is carried out according to methods known in the art, including but not limited to the steps of pre-freezing, primary drying and secondary drying. The skilled person understands that any method for removing water from the liquid formulation described in the present disclosure is applicable to the present disclosure.

[0142] The present disclosure also provides a lyophilized agent containing a bispecific anti-CD3 / CD20 polypeptide complex, which is prepared by the aforementioned method for preparing a lyophilized agent.

[0143] The present disclosure also provides a lyophilized agent containing a bispecific anti-CD3 / CD20 polypeptide complex, wherein the lyophilized agent can form the aforementioned liquid formulation after being reconstituted.

[0144] In some embodiments, the aforementioned lyophilized agent comprises:

[0145] (a) about 2 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0146] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0147] (c) about 0.0086 mmoL of sodium citrate,

[0148] (d) about 0.233 mmoL of trehalose, and

[0149] (e) about 0.2 mg of polysorbate 80;

[0150] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0151] The pH of the lyophilized agent is about 6.2.

[0152] In some embodiments, the aforementioned lyophilized agent comprises:

[0153] (a) about 2 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0154] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0155] (c) about 0.0086 mmoL of sodium citrate,

[0156] (d) about 0.2326 mmoL of trehalose, and

[0157] (e) about 0.2 mg of polysorbate 80;

[0158] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0159] The pH of the lyophilized agent is about 6.2.

[0160] In some embodiments, the aforementioned lyophilized agent comprises:

[0161] (a) about 2 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0162] (b) about 0.001 mmoL of citric acid,

[0163] (c) about 0.009 mmoL of sodium citrate,

[0164] (d) about 0.23 mmoL of trehalose, and

[0165] (e) about 0.2 mg of polysorbate 80;

[0166] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0167] The pH of the lyophilized agent is about 6.2.

[0168] In some embodiments, the aforementioned lyophilized agent comprises:

[0169] (a) about 5 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0170] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0171] (c) about 0.0086 mmoL of sodium citrate,

[0172] (d) about 0.233 mmoL of trehalose, and 6 mg of sodium chloride, and

[0173] (e) about 0.2 mg of polysorbate 80;

[0174] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0175] The pH of the lyophilized agent is about 6.

[0176] In some embodiments, the aforementioned lyophilized agent comprises:

[0177] (a) about 5 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0178] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0179] (c) about 0.0086 mmoL of sodium citrate,

[0180] (d) about 0.2326 mmoL of trehalose, and 6 mg of sodium chloride, and

[0181] (e) about 0.2 mg of polysorbate 80;

[0182] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0183] The pH of the lyophilized agent is about 6.

[0184] In some embodiments, the aforementioned lyophilized agent comprises:

[0185] (a) about 5 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0186] (b) about 0.001 mmoL of citric acid,

[0187] (c) about 0.009 mmoL of sodium citrate,

[0188] (d) about 0.23 mmoL of trehalose, and 6 mg of sodium chloride, and

[0189] (e) about 0.2 mg of polysorbate 80;

[0190] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0191] The pH of the lyophilized agent is about 6.

[0192] In some embodiments, the lyophilized agent is stable at 2-8°C for at least 1 month, at least 6 months, at least 12 months, at least 18 months or at least 24 months. In some embodiments, the lyophilized agent is stable at 25°C for at least 1 month, at least 6 months. In some embodiments, the lyophilized agent is stable at 40°C for at least 7 days, at least 14 days or at least 28 days. The present disclosure also provides a pharmaceutical composition containing a bispecific anti-CD3 / CD20 polypeptide complex, which is a solution obtained by reconstituting the aforementioned lyophilized agent.

[0193] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the step of re-dissolving the aforementioned lyophilized agent, wherein the solution used for re-dissolving includes but is not limited to water for injection, physiological saline or glucose solution, preferably water for injection.

[0194] The present disclosure also provides a product, which includes a container, wherein the container contains the liquid formulation of the present disclosure, or a lyophilized agent thereof, or a pharmaceutical composition reconstituted from a lyophilized agent.

[0195] In some embodiments, the aforementioned liquid formulation is stable at 2-8° C. for at least 1 month, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the aforementioned liquid formulation is stable at 40° C. for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 6 weeks.

[0196] The liquid formulation of the present invention, or its lyophilized agent, or the pharmaceutical composition reconstituted with the lyophilized agent can be administered intravenously (e.g., intravenous drip), intramuscularly, subcutaneously, parenterally, spinally or epidermally (e.g., by injection or push injection). The liquid formulation of the present invention, or its lyophilized agent, or the pharmaceutical composition reconstituted with the lyophilized agent can be diluted to a suitable concentration with a suitable diluent before administration to provide the best desired response (e.g., therapeutic response).

[0197] The present disclosure also provides use of the liquid formulation, or lyophilized agent, or pharmaceutical composition reconstituted from the lyophilized agent, or product described in the present disclosure in the preparation of a medicament for treating a CD20-related disease or condition.

[0198] In some embodiments, the CD20-related disease or condition is cancer, preferably, the cancer is selected from, but not limited to, lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer or gastric cancer.

[0199] In some embodiments, the CD20-associated disease or condition is a B-cell lymphoma, optionally a Hodgkin lymphoma or a non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), or Waldenstrom's macroglobulinemia (WM).

[0200] On the other hand, the present disclosure provides a pharmaceutical composition comprising a bispecific anti-CD3 / CD20 polypeptide complex, the pharmaceutical composition further comprising one or more of a buffer, an osmotic pressure regulator and / or a stabilizer, and / or a surfactant. In some embodiments, the pharmaceutical composition of the present disclosure is a lyophilized formulation comprising a bispecific anti-CD3 / CD20 polypeptide complex, a buffer, an osmotic pressure regulator and / or a stabilizer, and a surfactant.

[0201] In another aspect, the present disclosure provides a stable lyophilized formulation comprising a bispecific anti-CD3 / CD20 polypeptide complex and a buffer, wherein the buffer comprises one or more combinations of a histidine buffer, a succinate buffer, and a citrate buffer.

[0202] In some embodiments, the histidine salt buffer is histidine-histidine hydrochloride, the succinate buffer is succinic acid-sodium succinate, and the citrate buffer is citric acid-sodium citrate.

[0203] In some embodiments, the buffer in the aforementioned lyophilized formulation is a combination of one or more of histidine-histidine hydrochloride, succinic acid-sodium succinate, citric acid-sodium citrate buffer. In some embodiments, the buffer in the aforementioned lyophilized formulation is citric acid-sodium citrate buffer.

[0204] In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 0.1 mg / ml to about 50 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 0.5 mg / ml to about 20 mg / ml, for example, about 0.5 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 1 mg / ml to about 20 mg / ml, about 2 mg / ml to about 10 mg / ml, about 2 mg / ml to about 5 mg / ml, or about 2 mg / ml to about 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 2 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 5 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is about 10 mg / ml.

[0205] In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 0.1 mg / ml to 50 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 0.5 mg / ml to 20 mg / ml, for example, 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 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, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 1 mg / ml to 20 mg / ml, 2 mg / ml to 10 mg / ml, 2 mg / ml to 5 mg / ml, or 2 mg / ml to 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 2 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 3 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 5 mg / ml. In some embodiments, the concentration of the bispecific anti-CD3 / C20 polypeptide complex in the aforementioned lyophilized formulation is 10 mg / ml.

[0206] In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at about 5.0 to about 7.5. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at about 5.0 to about 6.5, for example, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at about 6.0 to about 6.5. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at about 6. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at about 6.2. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at about 6.5.

[0207] In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at 5.0 to 7.5. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at 5.0 to 6.5, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at 6.0 to 6.5. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at 6. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at 6.2. In some embodiments, the buffer in the aforementioned lyophilized formulation maintains the pH of the lyophilized formulation at 6.5.

[0208] In some embodiments, the concentration of the buffer in the aforementioned lyophilized formulation is about 1mM to about 30mM. In some embodiments, the concentration of the buffer in the aforementioned lyophilized formulation is about 5mM to about 20mM, for example, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM. In some embodiments, the concentration of the buffer in the aforementioned lyophilized formulation is about 10mM.

[0209] In some embodiments, the concentration of the buffer in the aforementioned lyophilized formulation is 1mM to 30mM. In some embodiments, the concentration of the buffer in the aforementioned lyophilized formulation is 5mM to 20mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM. In some embodiments, the concentration of the buffer in the aforementioned lyophilized formulation is 10mM.

[0210] In some embodiments, the pH of the aforementioned lyophilized formulation is about 5.0 to about 7.5. In some embodiments, the pH of the aforementioned lyophilized formulation is about 5.0 to about 6.5, for example, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5. In some embodiments, the pH of the aforementioned lyophilized formulation is about 6.0 to about 6.5. In some embodiments, the pH of the aforementioned lyophilized formulation is about 6. In some embodiments, the pH of the aforementioned lyophilized formulation is about 6.2. In some embodiments, the pH of the aforementioned lyophilized formulation is about 6.5.

[0211] In some embodiments, the pH of the aforementioned lyophilized formulation is 5.0 to 7.5. In some embodiments, the pH of the aforementioned lyophilized formulation is 5.0 to 6.5, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5. In some embodiments, the pH of the aforementioned lyophilized formulation is 6.0 to 6.5. In some embodiments, the pH of the aforementioned lyophilized formulation is 6. In some embodiments, the pH of the aforementioned lyophilized formulation is 6.2. In some embodiments, the pH of the aforementioned lyophilized formulation is 6.5.

[0212] In some embodiments, the aforementioned lyophilized formulation also includes an osmotic pressure regulator and / or a stabilizer. Wherein, the osmotic pressure regulator and / or a stabilizer may include sugar. The "sugar" used in the present disclosure may be a monosaccharide, a disaccharide, a trisaccharide, a polysaccharide, a sugar alcohol, a reducing sugar, and a non-reducing sugar, such as glucose, sucrose, trehalose, lactose, fructose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc. In some embodiments, the osmotic pressure regulator and / or a stabilizer also include one or more of sodium chloride, arginine-hydrochloride, glycine or proline. In some embodiments, the osmotic pressure regulator and / or a stabilizer in the aforementioned lyophilized formulation is a disaccharide, such as trehalose or sucrose. In some embodiments, the aforementioned freeze-dried formulation contains trehalose. In some embodiments, the aforementioned freeze-dried formulation contains trehalose, and one or more of sodium chloride, arginine-hydrochloride, glycine or proline. In some embodiments, the aforementioned freeze-dried formulation contains sucrose, and one or more of sodium chloride, arginine-hydrochloride, glycine or proline. In some embodiments, the aforementioned freeze-dried formulation contains trehalose and sodium chloride. In some embodiments, the aforementioned freeze-dried formulation contains trehalose and arginine-hydrochloride. In some embodiments, the aforementioned freeze-dried formulation contains sucrose and sodium chloride. In some embodiments, the aforementioned freeze-dried formulation contains sucrose and sodium chloride. In some embodiments, the aforementioned freeze-dried formulation contains sucrose and sodium chloride. In some embodiments, the aforementioned freeze-dried formulation contains sucrose and arginine-hydrochloride. In some embodiments, the aforementioned freeze-dried formulation contains sucrose and arginine-hydrochloride. In the formulation of the present application, sugar includes sugar and / or its hydrate, such as trehalose including trehalose and / or its hydrate, such as dihydrated trehalose.

[0213] In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 150mM to about 320mM. In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 180mM to about 265mM, for example, about 180mM, about 185mM, about 198mM, about 211mM, about 214mM, about 217mM, about 219mM, about 222mM, about 225mM, about 227mM, about 230mM, about 233mM, about 235mM, about 238mM, about 241mM, about 243mM, about 246mM, about 248mM, about 251mM, 254mM, about 256mM, about 259mM, about 262mM, about 264mM. In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 230mM to about 240mM, or about 230mM to about 235mM. In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 233mM. In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 234mM. In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 180mM to about 265mM, for example, about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39mM, about 222.03mM, about 224.67mM, about 227.31mM, about In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 232.6 mM. In some embodiments, the concentration of sugar in the aforementioned lyophilized formulation is about 233.7 mM.

[0214] In some embodiments, the concentration of trehalose in the aforementioned lyophilized formulation is about 150mM to about 320mM. In some embodiments, the concentration of trehalose in the aforementioned lyophilized formulation is about 180mM to about 265mM, for example, about 180mM, about 185mM, about 198mM, about 211mM, about 214mM, about 217mM, about 219mM, about 222mM, about 225mM, about 227mM, about 230mM, about 233mM, about 235mM, about 238mM, about 241mM, about 243mM, about 246mM, about 248mM, about 251mM, 254mM, about 256mM, about 259mM, about 262mM, about 264mM. In some embodiments, the concentration of trehalose in the aforementioned lyophilized formulation is about 230mM to about 240mM, or about 230mM to about 235mM. In some embodiments, the concentration of trehalose in the aforementioned lyophilized formulation is about 233mM. In some embodiments, the concentration of trehalose in the aforementioned lyophilized formulation is about 180mM to about 265mM, for example, about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39mM, about 222.03mM, about 224.67mM, about 227.31mM, About 229.96mM, about 232.6mM, about 235.24mM, about 237.89mM, about 240.53mM, about 243.17mM, about 245.82mM, about 248.46mM, about 251.1mM, 253.75mM, about 256.39mM, about 259.03mM, about 261.68mM, about 264.32mM. In some embodiments, the concentration of trehalose in the aforementioned lyophilized formulation is about 232.6mM.

[0215] In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned lyophilized formulation is about 10 mM to about 300 mM. In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned lyophilized formulation is about 20 mM to about 200 mM, for example, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned lyophilized formulation is about 50mM to about 150mM, about 100mM to about 110mM, or about 100mM to about 105mM. In some embodiments, the concentration of sodium chloride in the aforementioned lyophilized formulation is about 102.7mM. In some embodiments, the concentration of sodium chloride in the aforementioned lyophilized formulation is about 103mM. In some embodiments, the concentration of sodium chloride in the aforementioned lyophilized formulation is about 6 mg / ml. In some embodiments, the concentration of arginine-hydrochloride in the aforementioned lyophilized formulation is about 100 mM.

[0216] In some embodiments, the concentration of sodium chloride, arginine-HCl, glycine, or proline in the aforementioned lyophilized formulation is 10 mM to 300 mM. In some embodiments, the concentration of sodium chloride, arginine-HCl, glycine or proline in the aforementioned lyophilized formulation is 20 mM to 200 mM, for example, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM. In some embodiments, the concentration of sodium chloride, arginine-hydrochloride, glycine or proline in the aforementioned lyophilized formulation is 50mM to 150mM, 100mM to 110mM, or 100mM to 105mM. In some embodiments, the concentration of sodium chloride in the aforementioned lyophilized formulation is about 102.7mM. In some embodiments, the concentration of sodium chloride in the aforementioned lyophilized formulation is about 103mM. In some embodiments, the concentration of sodium chloride in the aforementioned lyophilized formulation is 6mg / ml. In some embodiments, the concentration of arginine-hydrochloride in the aforementioned lyophilized formulation is 100mM.

[0217] In some embodiments, the aforementioned lyophilized formulation further comprises a surfactant, which can be selected from polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-sweet Betaine, cetyl-betaine, lauroyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20. In some embodiments, the aforementioned lyophilized formulation comprises polysorbate 80 (also known as Tween 80).

[0218] In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is about 0.001% to about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is about 0.005% to about 0.08% (w / v), for example, about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.0 15% (w / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is about 0.01% to about 0.04% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is about 0.02% (w / v).

[0219] In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is 0.001% to 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is 0.005% to 0.08% (w / v), for example, 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), 0.01% (w / v), 0.015% (w / v), 0.02% (w / v), 0.025% (w / v), 0.03% (w / v), 0.035% (w / v), 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is 0.01% to 0.04% (w / v). In some embodiments, the concentration of polysorbate 80 in the aforementioned lyophilized formulation is 0.02% (w / v).

[0220] In some embodiments, the aforementioned lyophilized formulation comprises:

[0221] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0222] (b) about 1 mM to about 30 mM citrate buffer,

[0223] (c) about 150 mM to about 320 mM of a sugar, and optionally, one or more of about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and

[0224] (d) from about 0.001% to about 0.1% (w / v) of a surfactant,

[0225] The pH of the lyophilized formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0226] In some embodiments, the aforementioned lyophilized formulation comprises:

[0227] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0228] (b) about 1 mM to about 30 mM citrate buffer,

[0229] (c) about 150 mM to about 320 mM trehalose, and optionally, one or more of about 10 mM to about 300 mM sodium chloride, 10 mM to about 300 mM arginine-hydrochloride, 10 mM to about 300 mM glycine, or 10 mM to about 300 mM proline, and

[0230] (d) about 0.001% to about 0.1% (w / v) polysorbate 80,

[0231] The pH of the lyophilized formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0232] In some embodiments, the aforementioned lyophilized formulation comprises:

[0233] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0234] (b) about 5 mM to about 20 mM citrate buffer,

[0235] (c) about 180 mM to about 265 mM trehalose, and optionally, one or more of about 20 mM to about 200 mM sodium chloride, 20 mM to about 200 mM arginine-hydrochloride, 20 mM to about 200 mM glycine, or 20 mM to about 200 mM proline, and

[0236] (d) about 0.005% to about 0.08% (w / v) polysorbate 80,

[0237] wherein the pH of the lyophilized formulation is about 6.0 to about 6.5.

[0238] In some embodiments, the aforementioned lyophilized formulation comprises:

[0239] (a) about 0.5 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex (e.g., about 0.5 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml),

[0240] (b) about 5 mM to about 20 mM citric acid-sodium citrate buffer (e.g., about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM),

[0241] (c) about 180 mM to about 265 mM trehalose (e.g., about 180 mM, about 185 mM, about 198 mM, about 211 mM, about 214 mM, about 217 mM, about 219 mM, about 222 mM, about 225 mM, about 227 mM, about 230 mM, about 233 mM, about 235 mM, about 238 mM, about 241 mM) , about 243mM, about 246mM, about 248mM, about 251mM, 254mM, about 256mM, about 259mM, about 262mM, about 264mM; or about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39 mM, about 222.03mM, about 224.67mM, about 227.31mM, about 229.96mM, about 232.6mM, about 235.24mM, about 237.89mM, about 240.53mM, about 243.17mM, about 245.82mM, about 248.46mM, about 251.1mM, 253.75mM, about 256.39mM, about 259.03mM, about 261.68mM, about 264.32mM), and optionally, may further comprise one or more of about 20mM to about 200mM sodium chloride, 20mM to about 200mM arginine-hydrochloride, 20mM to about 200mM glycine or 20mM to about 200mM proline, and

[0242] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 (e.g., about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.015% (w / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v),

[0243] wherein the pH of the lyophilized formulation is about 6.0 to about 6.5 (eg, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5).

[0244] In some embodiments, the aforementioned lyophilized formulation comprises:

[0245] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0246] (b) about 1 mM to about 30 mM buffer (e.g., one or more of histidine-histidine hydrochloride buffer, succinic acid-sodium succinate buffer, citric acid-sodium citrate buffer),

[0247] (c) about 150 mM to about 320 mM of a sugar, and one or more of about 10 mM to about 300 mM sodium chloride, 10 mM to about 300 mM arginine-hydrochloride, 10 mM to about 300 mM glycine, or 10 mM to about 300 mM proline, and

[0248] (d) from about 0.001% to about 0.1% (w / v) of a surfactant,

[0249] The pH of the lyophilized formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0250] In some embodiments, the aforementioned lyophilized formulation comprises:

[0251] (a) about 0.5 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex (e.g., about 0.5 mg / ml, about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml),

[0252] (b) about 5 mM to about 20 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer (e.g., about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM),

[0253] (c) about 180 mM to about 265 mM trehalose or sucrose (e.g., about 180 mM, about 185 mM, about 198 mM, about 211 mM, about 214 mM, about 217 mM, about 219 mM, about 222 mM, about 225 mM, about 227 mM, about 230 mM, about 233 mM, about 235 mM, about 238 mM, about 241 mM, about 243 mM, about 246 mM, about 248 mM, about 251 mM, 254 mM, about 256 mM) or about 180mM, about 185.02mM, about 198.24mM, about 211.46mM, about 214.1mM, about 216.74mM, about 219.39mM, about 222.03mM, about 224.67mM, about 227.31mM, about 229.96mM, about 232.6mM, about 235.24mM, about 237.89mM, about 240.53mM, about about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 61 mM, about 62 mM, about 64 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 100 mM M, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 130mM, about 135mM, about 140mM, about 145mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM), and

[0254] (d) about 0.005% to about 0.08% (w / v) of polysorbate 80 or polysorbate 20 (e.g., about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.015% (w / v) / v), about 0.02% (w / v), about 0.025% (w / v), about 0.03% (w / v), about 0.035% (w / v), about 0.04% (w / v), about 0.045% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v)),

[0255] The pH of the lyophilized formulation is about 6.0 to about 6.5 (e.g., about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5). In some embodiments, the aforementioned lyophilized formulation comprises:

[0256] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0257] (b) about 1 mM to about 30 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer,

[0258] (c) about 150 mM to about 320 mM trehalose or sucrose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0259] (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20,

[0260] The pH of the lyophilized formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0261] In some embodiments, the aforementioned lyophilized formulation comprises:

[0262] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0263] (b) about 5 mM to about 20 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer,

[0264] (c) about 180 mM to about 265 mM trehalose or sucrose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0265] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20,

[0266] wherein the pH of the lyophilized formulation is about 6.0 to about 6.5.

[0267] In some embodiments, the aforementioned lyophilized formulation comprises:

[0268] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0269] (b) about 1 mM to about 30 mM citric acid-sodium citrate buffer,

[0270] (c) about 150 mM to about 320 mM trehalose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0271] (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20,

[0272] The pH of the lyophilized formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0273] In some embodiments, the aforementioned lyophilized formulation comprises:

[0274] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0275] (b) about 5 mM to about 20 mM citric acid-sodium citrate buffer,

[0276] (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and

[0277] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20,

[0278] wherein the pH of the lyophilized formulation is about 6.0 to about 6.5.

[0279] In some embodiments, the aforementioned lyophilized formulation comprises:

[0280] (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0281] (b) about 1 mM to about 30 mM citrate buffer,

[0282] (c) from about 150 mM to about 320 mM trehalose, and from about 10 mM to about 300 mM sodium chloride, and

[0283] (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20,

[0284] The pH of the lyophilized formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.5.

[0285] In some embodiments, the aforementioned lyophilized formulation comprises:

[0286] (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0287] (b) about 5 mM to about 20 mM citrate buffer,

[0288] (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride, and

[0289] (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20,

[0290] wherein the pH of the lyophilized formulation is about 6.0 to about 6.5.

[0291] In some embodiments, the aforementioned lyophilized formulation comprises:

[0292] (a) about 1 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0293] (b) about 5 mM to about 20 mM citrate buffer,

[0294] (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride, and

[0295] (d) about 0.01% to about 0.04% (w / v) polysorbate 80 or polysorbate 20,

[0296] wherein the pH of the lyophilized formulation is about 6.0 to about 6.5.

[0297] In some embodiments, the aforementioned lyophilized formulation comprises:

[0298] (a) about 2 mg / ml to about 3 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex,

[0299] (b) about 10 mM citric acid-sodium citrate buffer,

[0300] (c) about 233 mM trehalose, and

[0301] (d) about 0.02% (w / v) polysorbate 80,

[0302] wherein the pH of the lyophilized formulation is about 6.2.

[0303] In some embodiments, the aforementioned lyophilized formulation comprises:

[0304] (a) about 2 mg / ml to about 3 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0305] (b) about 10 mM citric acid-sodium citrate buffer,

[0306] (c) about 232.6 mM trehalose, and

[0307] (d) about 0.02% (w / v) polysorbate 80,

[0308] The pH of the lyophilized formulation is about 6.2.

[0309] In some embodiments, the aforementioned lyophilized formulation comprises:

[0310] (a) about 5 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0311] (b) about 10 mM citrate buffer,

[0312] (c) about 233 mM trehalose, and about 103 mM sodium chloride, and

[0313] (d) about 0.02% (w / v) polysorbate 80,

[0314] wherein the pH of the lyophilized formulation is about 6.

[0315] In some embodiments, the aforementioned lyophilized formulation comprises:

[0316] (a) about 5 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex,

[0317] (b) about 10 mM citrate buffer,

[0318] (c) about 232.6 mM trehalose, and about 102.7 mM sodium chloride, and

[0319] (d) about 0.02% (w / v) polysorbate 80,

[0320] wherein the pH of the lyophilized formulation is about 6.

[0321] In some embodiments, the aforementioned lyophilized formulation comprises:

[0322] (a) about 2 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0323] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0324] (c) about 0.0086 mmoL of sodium citrate,

[0325] (d) about 0.233 mmoL of trehalose, and

[0326] (e) about 0.2 mg of polysorbate 80;

[0327] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0328] The pH of the lyophilized formulation is about 6.2.

[0329] In some embodiments, the aforementioned lyophilized formulation comprises:

[0330] (a) about 2 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0331] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0332] (c) about 0.0086 mmoL of sodium citrate,

[0333] (d) about 0.2326 mmoL of trehalose, and

[0334] (e) about 0.2 mg of polysorbate 80;

[0335] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0336] The pH of the lyophilized formulation is about 6.2.

[0337] In some embodiments, the aforementioned lyophilized formulation comprises:

[0338] (a) about 2 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0339] (b) about 0.001 mmoL of citric acid,

[0340] (c) about 0.009 mmoL of sodium citrate,

[0341] (d) about 0.23 mmoL of trehalose, and

[0342] (e) about 0.2 mg of polysorbate 80;

[0343] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0344] The pH of the lyophilized formulation is about 6.2.

[0345] In some embodiments, the aforementioned lyophilized formulation comprises:

[0346] (a) about 5 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0347] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0348] (c) about 0.0086 mmoL of sodium citrate,

[0349] (d) about 0.233 mmoL of trehalose, and 6 mg of sodium chloride, and

[0350] (e) about 0.2 mg of polysorbate 80;

[0351] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0352] The pH of the lyophilized formulation is about 6.

[0353] In some embodiments, the aforementioned lyophilized formulation comprises:

[0354] (a) about 5 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0355] (b) about 0.0013 mmoL or about 0.0014 mmoL of citric acid,

[0356] (c) about 0.0086 mmoL of sodium citrate,

[0357] (d) about 0.2326 mmoL of trehalose, and 6 mg of sodium chloride, and

[0358] (e) about 0.2 mg of polysorbate 80;

[0359] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0360] The pH of the lyophilized formulation is about 6.

[0361] In some embodiments, the aforementioned lyophilized formulation comprises:

[0362] (a) about 5 mg of a bispecific anti-CD3 / CD20 polypeptide complex,

[0363] (b) about 0.001 mmoL of citric acid,

[0364] (c) about 0.009 mmoL of sodium citrate,

[0365] (d) about 0.23 mmoL of trehalose, and 6 mg of sodium chloride, and

[0366] (e) about 0.2 mg of polysorbate 80;

[0367] or a multiple of (a), (b), (c), (d) and (e), for example, an integer multiple or decimal multiple of the content of each component of (a), (b), (c), (d) and (e), or 1.5 times, 2 times, 3 times, 4 times the content of each component of (a), (b), (c), (d) and (e), or an integer multiple of 5 or more of the content of each component of (a), (b), (c), (d) and (e),

[0368] The pH of the lyophilized formulation is about 6.

[0369] In some embodiments, the lyophilized formulation is stable at 2-8° C. for at least 1 month, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 25° C. for at least 1 month, at least 6 months. In some embodiments, the lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

[0370] The present disclosure also provides a pharmaceutical composition containing a bispecific anti-CD3 / CD20 polypeptide complex, which is a solution obtained by reconstituting the aforementioned lyophilized formulation.

[0371] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the step of re-dissolving the aforementioned lyophilized formulation, wherein the solution used for re-dissolving includes but is not limited to water for injection, physiological saline or glucose solution, preferably water for injection.

[0372] The present disclosure also provides an article of manufacture, which includes a container containing the lyophilized formulation of the present disclosure, or a reconstituted pharmaceutical composition thereof.

[0373] The lyophilized formulation of the present invention, or its reconstituted pharmaceutical composition, can be administered intravenously (e.g., intravenous drip), intramuscularly, subcutaneously, parenterally, spinally, or epidermally (e.g., by injection or bolus). The lyophilized formulation of the present invention, or its reconstituted pharmaceutical composition, can be diluted to a suitable concentration with a suitable diluent before administration to provide the best desired response (e.g., therapeutic response).

[0374] In another aspect, the present disclosure provides use of the lyophilized formulation of the present disclosure, or a reconstituted pharmaceutical composition or product thereof, in the preparation of a medicament for treating a CD20-related disease or condition.

[0375] In some embodiments, the CD20-related disease or condition is cancer, preferably, the cancer is selected from, but not limited to, lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer or gastric cancer.

[0376] In some embodiments, the CD20-associated disease or condition is a B-cell lymphoma, optionally a Hodgkin lymphoma or a non-Hodgkin lymphoma, wherein the non-Hodgkin lymphoma includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), or Waldenstrom's macroglobulinemia (WM).

[0377] In an alternative embodiment, the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or the bispecific anti-CD3 / CD20 polypeptide complex in the article of manufacture comprises a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

[0378] The first antigen binding moiety comprises:

[0379] a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and

[0380] a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2),

[0381] wherein C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain bond between C1 and C2, and the non-natural interchain bond is capable of stabilizing the dimer, and

[0382] The second antigen binding moiety comprises:

[0383] a second heavy chain variable domain (VH2) of a second antibody, operably linked to the antibody heavy chain CH1 domain, and

[0384] a second light chain variable domain (VL2) of a second antibody operably linked to an antibody light chain constant (CL) domain,

[0385] wherein one of said first and said second antigen binding moiety is an anti-CD3 binding moiety and the other is an anti-CD20 binding moiety,

[0386] The anti-CD3 binding portion is derived from an anti-CD3 antibody and comprises:

[0387] a) a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 13,

[0388] b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 14,

[0389] c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 15,

[0390] d) a kappa light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 16,

[0391] e) a kappa light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 17, and

[0392] f) a kappa light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 18,

[0393] The anti-CD20 binding portion is derived from an anti-CD20 antibody, which comprises:

[0394] a) a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 19,

[0395] b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 20,

[0396] c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 21,

[0397] d) a kappa light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 22,

[0398] e) a kappa light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 23, and

[0399] f) a kappa light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 24.

[0400] In some embodiments, the anti-CD3 binding portion is derived from an anti-CD3 antibody comprising:

[0401] a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0402] b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14,

[0403] c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15,

[0404] d) a kappa light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16,

[0405] e) a kappa light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and

[0406] f) a kappa light chain CDR3 comprising the amino acid sequence of SEQ ID NO:18.

[0407] In some embodiments, the anti-CD20 binding portion is derived from an anti-CD20 antibody comprising:

[0408] a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 19,

[0409] b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20,

[0410] c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 21,

[0411] d) a kappa light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22,

[0412] e) a kappa light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and

[0413] f) a kappa light chain CDR3 comprising the amino acid sequence of SEQ ID NO:24.

[0414] In an alternative embodiment, the anti-CD3 binding portion of the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product is derived from an anti-CD3 antibody, which comprises a heavy chain variable domain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 27, and a light chain variable domain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 28. In some embodiments, the anti-CD3 binding portion is derived from an anti-CD3 antibody, which comprises a heavy chain variable domain sequence comprising an amino acid sequence variant selected from the group consisting of SEQ ID NOs: 25 and 27, for example, a heavy chain variable domain sequence comprising an amino acid sequence with amino acid SS deleted at the C-terminus of SEQ ID NOs: 25 or 27, and a light chain variable domain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 28. In some embodiments, the anti-CD3 binding portion is derived from an anti-CD3 antibody comprising a heavy chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-CD3 binding portion is derived from an anti-CD3 antibody comprising a heavy chain variable domain sequence comprising an amino acid sequence variant of SEQ ID NO: 27, e.g., a heavy chain variable domain sequence having an amino acid sequence with amino acid SS deleted at the C-terminus of SEQ ID NO: 27, and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 28.

[0415] In an alternative embodiment, the anti-CD20 binding portion of the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product is derived from an anti-CD20 antibody comprising a heavy chain variable domain sequence comprising the amino acid sequence of SEQ ID NOs: 29 and 31 and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NOs: 30 and 32. In some embodiments, the anti-CD20 binding portion is derived from an anti-CD20 antibody comprising a heavy chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 32.

[0416] In an optional embodiment, the first antigen binding moiety is connected to a first dimerization domain, and the second antigen binding moiety is connected to a second dimerization domain, wherein the first and second dimerization domains are associated. In an optional embodiment, the association is achieved via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof.

[0417] In alternative embodiments, said first and / or said second dimerization domain comprises at least a portion of an antibody hinge region, which is optionally derived from IgG1, IgG2 or IgG4.

[0418] In an alternative embodiment, the first and / or the second dimerization domain comprises an antibody CH2 domain, and / or an antibody CH3 domain.

[0419] In an alternative embodiment, the first dimerization domain is operably linked to the first TCR constant region (C1) at a third engagement domain.

[0420] In alternative embodiments, the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen binding moiety.

[0421] In alternative embodiments, said first and said second dimerization domains are different and are associated in a manner that disfavors homodimerization and / or favors heterodimerization.

[0422] In alternative embodiments, said first and said second dimerization domains are capable of associating as heterodimers via handles into holes, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

[0423] In an alternative embodiment, the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product comprises a combination of the following four polypeptide sequences: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

[0424] In an alternative embodiment, the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product comprises a combination of the following four polypeptide sequences: SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.

[0425] In an alternative embodiment, the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product comprises a combination of the following four polypeptide sequences: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.

[0426] In an alternative embodiment, the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product comprises a combination of the following four polypeptide sequences: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48.

[0427] In an alternative embodiment, the bispecific anti-CD3 / CD20 polypeptide complex in the aforementioned liquid formulation, or lyophilized agent or its reconstituted pharmaceutical composition, or lyophilized formulation or its reconstituted pharmaceutical composition, or product comprises a combination of the following four polypeptide sequences: SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52.

[0428] In an alternative embodiment, one or more amino acids at positions 193, 182, 203, 206 or 207 of the polypeptide shown in SEQ ID NO: 44 are modified to any amino acid except serine and / or threonine, thereby eliminating the glycosylation site, preferably, the amino acid at position 193 is modified. In an alternative embodiment, the amino acid at position 193 of the polypeptide shown in SEQ ID NO: 44 is modified to alanine, glycine, proline or valine.

[0429] The foregoing and other features and advantages of the present disclosure will become apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0430] Figure 1 Schematic diagrams of the antibody formats studied are presented, wherein "E17R-1", "F16-1" and "F17R-1" schematically represent the formats of bispecific antibodies W3278-T2U3.E17R-1.uIgG4.SP, W3278-T3U2.F16-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP, respectively, and "F18R-1" schematically represents the formats of bispecific antibodies W3278-U2T3.F18R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP. In the context of the present disclosure, "U" in the bispecific antibody nomenclature refers to an anti-CD20 antibody or an anti-CD20 binding portion, and "T" in the bispecific antibody nomenclature refers to an anti-CD3 antibody or an anti-CD3 binding portion. The constant regions (CL and CH1) of "T" were replaced by the constant domains of TCR to design a unique light-heavy chain interface orthogonal to conventional antibodies. The TCR-modified "T" and the native "U" were used together with the "handle into hole" mutation in the Fc domain to design the bispecific antibody formats "E17R-1", "F16-1", "F17R-1" and "F18R-1".

[0431] Figure 2 Target binding detected by FACS is shown: (A) Binding detection of the bispecific antibody (WBP3278 BsAb) of the present disclosure to Jurkat cells, (B) Binding detection of the bispecific antibody (WBP3278 BsAb) of the present disclosure to Raji cells.

[0432] Figure 3 Simultaneous dual target binding detected by FACS is shown.

[0433] Figure 4 T cell killing is shown.

[0434] Figure 5A and 5B T cell activation as indicated by CD25 expression and CD69 expression are shown, respectively.

[0435] Figure 6 CD4 + IL-2 (A) and TNF-α (B) release by T cells.

[0436] Figure 7 Results for serum stability are shown.

[0437] Figure 8 The BsAbs of the present disclosure were shown to have dose-dependent anti-tumor activity in an in vivo therapeutic treatment model.

[0438] Fig. 9 Depletion of peripheral blood circulating B cells following administration of WBP3278 lead Ab (ie, W3278-U2T3.F18R-1.uIgG4) to naive male cynomolgus monkeys is shown.

[0439] Fig. 10A , 10B and 10C show changes in peripheral blood circulating T cell levels after treatment with WBP3278 lead Ab.

[0440] Fig.11 Shown are changes in circulating cytokine levels following treatment with WBP3278 lead Ab.

[0441] Fig.12 Shown are the changes in serum concentrations of WBP3278 lead Ab over time. DETAILED DESCRIPTION

[0442] The following description of the present disclosure is merely for the purpose of illustrating various embodiments of the present disclosure.

[0443] definition

[0444] "Histidine salt buffer" is a buffer containing histidine ions, or a buffer containing histidine and / or histidine salts, wherein histidine includes histidine and / or its hydrates, and histidine salts include histidine salts and / or its hydrates. Examples of histidine salt buffers include buffers such as histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, histidine-histidine hydrochloride, and histidine-histidine hydrochloride, preferably histidine-histidine hydrochloride. Histidine-histidine hydrochloride refers to a combination of histidine and histidine hydrochloride, wherein histidine includes histidine and / or its hydrates, and histidine hydrochloride includes histidine hydrochloride and / or its hydrates, and in one example, a histidine-histidine hydrochloride buffer is prepared from histidine and histidine hydrochloride monohydrate (C6H9N3O2·HCl·H2O).

[0445] "Succinate buffer" is a buffer comprising succinate ions, or a buffer comprising succinic acid and / or succinate, wherein succinic acid includes succinic acid and / or its hydrates, and succinate includes succinate and / or its hydrates. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like, and a preferred succinate buffer is succinic acid-sodium succinate. Succinic acid-sodium succinate refers to a combination of succinic acid and sodium succinate, wherein succinic acid includes succinic acid and / or its hydrates, and sodium succinate includes sodium succinate and / or its hydrates.

[0446] "Citrate buffer" is a buffer comprising citrate ions, or a buffer comprising citric acid and / or citrate, wherein citric acid includes citric acid and / or its hydrate, and citrate includes citrate and / or its hydrate. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc., and a preferred citrate buffer is citric acid-sodium citrate. Citric acid-sodium citrate refers to a combination of citric acid and sodium citrate, wherein citric acid includes citric acid and / or its hydrate, and sodium citrate includes sodium citrate and / or its hydrate. In one example, citric acid-sodium citrate buffer is prepared by citric acid monohydrate (C6H8O7·H2O) and sodium citrate dihydrate (C6H5 Na3O7·2H2O).

[0447] "Buffer" and "buffer" are buffers that help maintain the pH of the formulation in a range close to physiological conditions. Buffers / agents suitable for use in the present disclosure include pairs of inorganic acids and their salts or organic acids and their salts, such as citrate buffers / agents (e.g., citric acid-potassium citrate mixtures, or citric acid-sodium citrate mixtures, etc.), succinate buffers / agents (e.g., succinic acid-sodium succinate mixtures, or succinic acid-sodium hydroxide mixtures, etc.), histidine salt buffers / agents (e.g., histidine-histidine hydrochloride mixtures). In one example, citric acid-sodium citrate buffers / agents are prepared from citric acid monohydrate (C6H8O7·H2O) and sodium citrate dihydrate (C6H5 Na3O7·2H2O).

[0448] "Pharmaceutical composition" and "formulation" are intended to encompass a product comprising the specified ingredients (e.g., a bispecific anti-CD3 / CD20 polypeptide complex) in optionally specified amounts, as well as any product produced directly or indirectly by combining the specified ingredients in optionally specified amounts. A "stable" or "stabilized" pharmaceutical composition or formulation is one in which the active ingredient (e.g., a protein or antibody) substantially retains its physical stability and / or chemical stability and / or biological activity when stored therein. A variety of analytical techniques for measuring the stability of active ingredients are known in the art, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and other storage conditions for a selected period of time. For example, if the active ingredient does not show a significant increase in aggregation, precipitation and / or denaturation after visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and differential scanning calorimetry (DSC), then the active ingredient "retains its physical stability" in the pharmaceutical formulation. Preferably, when the formulations of the present disclosure are used, 5% or less, 4% or less, preferably 3% or less of the active ingredient forms aggregates, such as by SEC-HPLC or any other suitable method for measuring aggregate formation. If the active ingredient (e.g., protein or antibody) does not show significant chemical changes, then the active ingredient (e.g., protein or antibody) "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein or antibody. Degradation processes that often change the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement, etc.) and isomerization (evaluated by measuring isoaspartate content, peptide mapping, etc.). If the biological activity of the active ingredient (e.g., protein or antibody) at a given time is at least about 90% (within the measurement error) of the biological activity when the pharmaceutical formulation is prepared, then the active ingredient (e.g., protein or antibody) "retains its biological activity" at a given time in the pharmaceutical formulation, for example, as determined by an antigen binding assay. In some embodiments, the pharmaceutical composition or the formulation is a water-soluble injection, which includes but is not limited to a water-soluble formulation that has not been lyophilized or a water-soluble formulation reconstituted from a lyophilized powder.In other embodiments, the pharmaceutical composition or the formulation is a lyophilized preparation. The lyophilized preparation refers to an aqueous solution undergoing a lyophilization process to prepare a preparation, and lyophilization is a stabilization process in which a substance is first frozen, and then the amount of solvent is first reduced by sublimation (primary drying process), and then the amount of solvent is reduced by desorption (secondary drying process) until the amount of solvent is a value that no longer supports biological activity or chemical reaction. The lyophilized preparation of the present disclosure can also be dried by other methods known in the art, such as spray drying and bubbling drying (bubbledrying).

[0449] The articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "a formulation" refers to one formulation or more than one formulation.

[0450] The term "about" or "approximately" refers to a numerical value within an acceptable error range of a specific value determined by a person of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in the art, "about" or "approximately" can mean a standard deviation within or exceeding 1. Or "about" or "approximately" or "means a range of plus or minus 15%, 10%, 5%, or 1% of the stated value. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise specified in the present disclosure, "about XX" or "approximately XX" or "substantially comprising XX" means a numerical value within an acceptable error range of the specific value "XX" (including the numerical value "XX" itself, as well as a numerical value within an acceptable error range of the numerical value determined by a person of ordinary skill in the art).

[0451] Throughout this disclosure, unless the context dictates otherwise, the words "comprise," "include," and "contain" will be understood to mean including the stated step or element or set of steps or elements, but not excluding any other step or element or set of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or necessary, and no other elements may be present. "Consisting essentially of" means including any element listed after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as described in detail in this disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are required or necessary, but other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements.

[0452] References to "one embodiment", "an embodiment", "a specific embodiment", "a related embodiment", "a certain embodiment", "another embodiment" or "a further embodiment" or combinations thereof throughout this disclosure indicate that the specific features, structures or characteristics described in connection with the embodiment are included in at least one embodiment of the disclosure. Therefore, the appearance of the aforementioned terms throughout this specification does not necessarily refer to the same embodiment. In addition, the specific features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

[0453] The terms "polypeptide", "peptide" and "protein" are used interchangeably in the present disclosure and refer to a polymer of amino acid residues, or an aggregate of a plurality of amino acid residue polymers. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids (i.e., α carbon is bound to hydrogen, carboxyl, amino and R groups), such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. α-carbon refers to the first carbon atom attached to a functional group (such as a carbonyl group). The beta carbon refers to the second carbon atom attached to the alpha carbon, and this system continues to name carbon atoms in alphabetical order of the Greek alphabet. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but act in a manner similar to naturally occurring amino acids. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. Polypeptide sequences are generally described as the left-hand end of the polypeptide sequence being the amino terminus (N-terminus); the right-hand end of the polypeptide sequence being the carboxyl terminus (C-terminus). A "polypeptide complex" as used in the present disclosure refers to a complex comprising one or more polypeptides associated with achieving certain functions. In certain embodiments, the polypeptide is immunologically relevant.

[0454] The term "antibody" as used in the present disclosure encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that can bind to a specific antigen. A natural complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and the first, second and third constant regions (CH1, CH2, CH3, respectively), while each light chain consists of a variable region ("LCVR") and a constant region (CL). The heavy chains of mammals can be divided into α, δ, ε, γ and μ, and the light chains of mammals can be divided into λ or κ. The antibody is "Y"-shaped, and the backbone of the "Y"-shaped structure is composed of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y"-shaped structure contains the variable region and the first constant region of one of the heavy chains, which is combined with the variable region and constant region of one light chain. The variable regions of the light chain and the heavy chain are responsible for the binding of the antigen. The variable region of each chain contains three hypervariable loops, called complementarity determining regions (CDRs) (the CDRs of the light (L) chain include LCDR1, LCDR2, LCDR3, and the CDRs of the heavy (H) chain include HCDR1, HCDR2, HCDR3). The CDR boundaries of an antibody may be named or identified by the Kabat, Chothia, or Al-Lazikani nomenclature (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). Among them, the three CDRs are separated by flanking continuous parts called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. HCVR and LCVR each contain 4 FRs, and CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding, but have various effector functions. Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. Depending on whether they contain α, δ, ε, γ and μ heavy chains, antibodies can be divided into five major classifications or isotypes: IgA, IgD, IgE, IgG and IgM.Several major antibody classifications can be further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain), etc.

[0455] The term "variable domain" used in the present disclosure, when used for antibodies, refers to an antibody variable region or fragment thereof comprising one or more CDRs. Although a variable domain may comprise a complete variable region (such as a HCVR or LCVR), it may also comprise less than a complete variable region but still retain the ability to bind to an antigen or form an antigen binding site.

[0456] The term "antigen binding portion" used in the present disclosure refers to an antibody fragment formed by an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen binding portions include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding portion may bind to the same antigen as the parent antibody. In certain embodiments, the antigen binding portion may comprise one or more CDRs from a specific human antibody grafted to a framework region from one or more different human antibodies. More detailed formats of antigen binding portions are described in Spiess et al., 2015 (supra) and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), the entire contents of which are incorporated herein by reference.

[0457] The "Fab" of an antibody refers to the part of the antibody that is bound together by a light chain (comprising a variable region and a constant region) and a variable region and a first constant region of a heavy chain via a disulfide bond. In certain embodiments, the constant regions of both the light chain and the heavy chain are replaced by TCR constant regions.

[0458] "Fab'" refers to the Fab fragment including part of the hinge region.

[0459] "F(ab')2" refers to a dimer of Fab.

[0460] The "fragment difficult (Fd)" of an antibody refers to the amino-terminal half of the heavy chain fragment that can combine with the light chain to form the Fab.

[0461] The "Fc" of an antibody refers to the part of the antibody consisting of the second (CH2) and third (CH3) constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of an antibody is responsible for a variety of different effector functions, such as ADCC and CDC, but does not participate in antigen binding.

[0462] The "hinge region" with respect to antibodies comprises the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen binding regions to move independently.

[0463] The term "CH2 domain" as used in the present disclosure refers to a portion comprising a heavy chain molecule extending from, for example, about amino acid 244 to amino acid 360 of an IgG antibody, using the conventional numbering scheme (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E. et al., US Department of Health and Human Services (1983)).

[0464] The "CH3 domain" extends from the CH2 domain of the IgG molecule to the C-terminus and comprises approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, further comprise a CH4 region.

[0465] The "Fv" of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of one light chain combined with the variable region of one heavy chain. Several Fv designs are provided, including dsFv, in which the connection between the two domains is enhanced by an introduced disulfide bond; and a peptide linker can be used to bind the two domains together into a single polypeptide to form an scFv. Fv constructs containing the variable domains of a heavy immunoglobulin chain or a light immunoglobulin chain connected to the variable domains and constant domains of the corresponding immunoglobulin heavy or light chain have been produced. Fv has also been multimerized to form bifunctional antibodies and trifunctional antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0466] "ScFab" refers to a fusion polypeptide with the Fd linked to the light chain via a polypeptide linker, resulting in a single-chain Fab fragment (scFab).

[0467] "TriFab" refers to a trivalent, bispecific fusion protein composed of three units with Fab functionality. TriFab contains two common Fabs fused to an asymmetric Fab-like part.

[0468] "Fab-Fab" refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm.

[0469] "Fab-Fv" refers to a fusion protein formed by fusing HCVR to the C-terminus of the Fd chain, and LCVR to the C-terminus of the light chain. "Fab-dsFv" molecules can be formed by introducing an interdomain disulfide bond between the HCVR domain and the LCVR domain.

[0470] "MAb-Fv" or "IgG-Fv" refers to a fusion protein formed by fusion of the HCVR domain to the C-terminus of one Fc chain and the LCVR domain expressed alone or fused to the C-terminus of the other Fc chain, thereby generating a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, the Fv of which is stabilized by an interdomain disulfide bond.

[0471] "ScFab-Fc-scFv2" and "ScFab-Fc-scFv" refer to fusion proteins formed by the fusion of single-chain Fab with Fc and disulfide-stabilized Fv domains.

[0472] "Attached IgG" refers to a fusion protein in which the Fab arm is fused to IgG to form a bispecific (Fab)2-Fc. It can form an "IgG-Fab" or "Fab-IgG" in which the Fab is fused to the C-terminus or N-terminus of the IgG molecule, regardless of whether a linker is present. In some embodiments, the attached IgG can be further modified to the form of IgG-Fab4 (see Brinkman et al., 2017, supra).

[0473] "DVD-Ig" refers to a dual variable domain antibody formed by fusing additional HCVR domains and LCVR domains of a second specificity to IgG heavy and light chains. "DVD-Ig" refers to a related format in which two HCVR and two LCVR domains are linked in a manner that allows cross-pairing of the variable HCVR-LCVR domains, which are arranged (from N to C terminus) in the order of HCVRA-HCVRB and LCVRB-LCVRA, or in the order of HCVRB-HCVRA and LCVRA-LCVRB.

[0474] "CrossMab" refers to the technology of pairing an unmodified light chain with a corresponding unmodified heavy chain, and pairing a modified light chain with a corresponding modified heavy chain, thereby generating antibodies with reduced mispairing in the light chain.

[0475] "BiTE" is a bispecific T-cell engagement molecule comprising a first scFv with specificity for a first antigen in a LCVR-HCVR orientation linked to a second scFv with specificity for a second antigen in a HCVR-LCVR orientation.

[0476] "WuXiBody" is a bispecific antibody comprising a soluble chimeric protein having the variable domains of an antibody and the constant domains of a TCR, wherein the subunits of the TCR constant domains (e.g., the α and β domains) are connected by engineered disulfide bonds.

[0477] When "percent sequence identity" is used for amino acid sequences (or nucleic acid sequences), it refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after the sequences are aligned and, if necessary, intervals are introduced to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid (or nucleic acid) sequences by tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tools or can appropriately adjust the parameters according to the needs of the comparison, such as by selecting a suitable algorithm.

[0478] "Antigen" or "Ag" used in the present disclosure refers to a compound, composition, peptide, polypeptide, protein or substance that can stimulate the production of antibodies or T cell responses in cell culture or in animals, including compositions added to cell cultures (such as hybridomas), or injected or absorbed into animals (such as compositions containing cancer-specific proteins). Antigens react with specific humoral or cellular immune products (such as antibodies, including products induced by heterologous antigens).

[0479] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent (such as an antibody) binds. An epitope can be formed from consecutive amino acids (also called linear or sequential epitopes) or non-continuous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitopes). Epitopes formed from consecutive amino acids are usually arranged linearly along the primary amino acid residues on the protein, and small stretches of consecutive amino acids can be digested by antigens bound to major histocompatibility complex (MHC) molecules, or retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. An epitope usually contains at least 3, more usually at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation.

[0480] "Specific binding" or "specific binding" in the present disclosure refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure specifically binds to the antigen, and its binding affinity (K D )≤10 -6 M (e.g. ≤5×10 -7 M, ≤2×10 -7 M, ≤10 - 7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤2×10 -9 M, ≤10 -9 M or ≤10 -10 M). K used in the present disclosure D The ratio of the dissociation rate to the association rate (k off / k on ), can be determined by using surface plasmon resonance, for example using an instrument such as Biacore.

[0481] The term "operably linked" or "operably connected" refers to the juxtaposition of two or more biological sequences of interest in a manner that places them in a relationship that allows them to act in a desired manner, regardless of the presence of spacers or linkers. When applied to polypeptides, the term is intended to indicate that the polypeptide sequences are connected in a manner that allows the connected product to have the desired biological function. For example, an antibody variable region can be operably linked to a constant region to form a stable product with antigen binding activity. The term can also be applied to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), the term is intended to indicate that the polynucleotide sequences are connected in a manner that allows the expression of the polypeptide to be regulated by the polynucleotide.

[0482] The term "fusion" or "fused" when used for amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences into a non-naturally occurring single amino acid sequence, for example, by chemical bonding or recombinant means. The fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0483] The term "spacer" used in the present disclosure refers to an artificial amino acid sequence connected by peptide bonds and used to connect one or more polypeptides, having 1, 2, 3, 4 or 5 amino acid residues, or a length of 5 to 15, 20, 30, 50 or more amino acid residues. The spacer may or may not have a secondary structure. Spacer sequences are well known in the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994). Any suitable spacer known in the art can be used.

[0484] The term "antigen specificity" refers to a particular antigen or epitope thereof that is selectively recognized by an antigen binding molecule.

[0485] The term "substitution" used in the present disclosure when used for amino acid residues refers to the substitution of one or more naturally occurring or introduced amino acids with another in a peptide, polypeptide or protein. Substitution in a polypeptide may result in a decrease, increase or elimination of polypeptide function.

[0486] Substitutions may also be "conservative substitutions", which, when directed to an amino acid sequence, refer to the substitution of an amino acid residue with another different amino acid residue having a side chain of similar physicochemical properties, or substitution of amino acids that are not essential to the activity of the polypeptide. For example, conservative substitutions may be made between non-polar side chain amino acid residues (e.g., Met, Ala, Val, Leu and Ile, Pro, Phe, Trp), between uncharged polar side chain residues (e.g., Cys, Ser, Thr, Asn, Gly and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys and Arg), between β-branched side chain amino acids (e.g., Thr, Val and Ile), between sulfur-containing side chain amino acids (e.g., Cys and Met), or between aromatic side chain residues (e.g., Trp, Tyr, His and Phe). In certain embodiments, substitutions, deletions or additions may also be considered "conservative substitutions". The number of inserted or deleted amino acids may range from about 1 to 5. Conservative substitutions usually do not cause significant changes in the conformational structure of the protein and thus can maintain the biological activity of the protein.

[0487] As used in the present disclosure, the term "mutation" or "mutated" when applied to an amino acid residue refers to a substitution, insertion or addition of an amino acid residue.

[0488] The term "T cell" as used in the present disclosure refers to a type of lymphocyte that plays an important role in cell-mediated immunity, including helper T cells (such as CD4 + T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 3 T cells, T helper type 17 T cells), cytotoxic T cells (such as CD8 + T cells), memory T cells (e.g., central memory T cells (TCM cells), effector memory T cells (TEM cells and TEMRA cells), and resident memory T cells (TRM), which are CD8 + or CD4 + ), natural killer T (NKT) cells and suppressor T cells.

[0489] The natural "T cell receptor" or natural "TCR" is a heterodimeric T cell surface protein that binds to the invariant CD3 chain to form a complex capable of mediating signal transduction. The TCR belongs to the immunoglobulin superfamily and is similar to a half antibody with a single heavy chain and a single light chain. The natural TCR has an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a membrane-proximal constant region and a membrane-distal variable region.

[0490] The term "subject" or "individual" or "animal" or "patient" used in the present disclosure refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, alleviation, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, livestock animals, farm animals, and zoo, competitive or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0491] "Treatment" or "therapy" includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof. For cancer, "treat" or "therapy" may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treat" or "therapy" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0492] Bispecific anti-CD3 / CD20 peptide complex

[0493] The term "bispecific" used in the present disclosure means that there are two antigen binding moieties, each of which can specifically bind to different antigens or different epitopes on the same antigen. The bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure comprises a first antigen binding moiety associated with a second antigen binding moiety, and one of which specifically binds to CD3 and the other specifically binds to CD20. That is, the first antigen binding moiety can specifically bind to CD3, and the second antigen binding moiety can specifically bind to CD20. Alternatively, the first antigen binding moiety can specifically bind to CD20, and the second antigen binding moiety can specifically bind to CD3. In the present disclosure, the term "bispecific anti-CD3xCD20 polypeptide complex" is used interchangeably with "polypeptide complex targeting CD3 and CD20" or "anti-CD3 and CD20 polypeptide complex" or "bispecific anti-CD3 / CD20 polypeptide complex".

[0494] In certain embodiments, the present disclosure provides a bispecific anti-CD3 / CD20 polypeptide complex comprising a first antigen binding moiety associated with a second antigen binding moiety, wherein:

[0495] The first antigen binding moiety comprises:

[0496] a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and

[0497] a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2),

[0498] wherein: C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain bond between C1 and C2, and the non-natural interchain bond is capable of stabilizing the dimer, and

[0499] The second antigen binding moiety comprises:

[0500] a second heavy chain variable domain (VH2) of a second antibody, operably linked to the antibody heavy chain CH1 domain, and

[0501] a second light chain variable domain (VL2) of a second antibody operably linked to an antibody light chain constant (CL) domain,

[0502] wherein: one of said first and said second antigen binding moiety is an anti-CD3 binding moiety and the other is an anti-CD20 binding moiety,

[0503] The anti-CD3 binding portion is derived from an anti-CD3 antibody and comprises:

[0504] a) a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 13,

[0505] b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 14,

[0506] c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 15,

[0507] d) a kappa light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 16,

[0508] e) a kappa light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 17, and

[0509] f) a kappa light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 18,

[0510] The anti-CD20 binding portion is derived from an anti-CD20 antibody, which comprises:

[0511] a) a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 19,

[0512] b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 20,

[0513] c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 21,

[0514] d) a kappa light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 22,

[0515] e) a kappa light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 23, and

[0516] f) a kappa light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 24.

[0517] In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure comprises a first antigen binding moiety comprising a sequence derived from a TCR constant region, but the second antigen binding moiety does not comprise a sequence derived from a TCR constant region.

[0518] The bispecific anti-CD3 / CD20 polypeptide complexes provided by the present disclosure are significantly less prone to mispairing of heavy and light chain variable domains. Without wishing to be bound by any theory, it is believed that the stable TCR constant regions in the first antigen binding moiety can specifically bind to each other and thus facilitate highly specific pairing of target VH1 and VL1, while hindering VH1 or VL1 from forming unwanted mispairing with other variable regions that do not provide target antigen binding sites.

[0519] In certain embodiments, the second antigen binding moiety further comprises an antibody constant CH1 domain operably linked to VH2, and an antibody light chain constant domain operably linked to VL2. Thus, the second antigen binding moiety comprises Fab.

[0520] When the first, second, third and fourth variable domains (e.g., VH1, VH2, VL1 and VL2) are expressed in a cell, it is highly desirable that VH1 is specifically paired with VL1 and VH2 is specifically paired with VL2 so that the resulting bispecific protein product will have the correct antigen binding specificity. However, in the prior art such as hybrid-hybridoma (or quadroma), VH1, VH2, VL1 and VL2 are randomly paired, resulting in the generation of up to 10 different molecules, of which only one is a functional bispecific antigen binding molecule. This not only reduces the yield, but also complicates the purification of the target product.

[0521] Compared to the case where both the first and second antigen binding moieties are the corresponding parts of natural Fab, the bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure is superior because the variable domains are less prone to mispairing. In an exemplary example, the first antigen binding domain comprises VH1-C1 paired with VL1-C2, and the second antigen binding domain comprises VH2-CH1 paired with VL2-CL. It has been unexpectedly found that C1 and C2 preferentially associate with each other and are less likely to associate with CL or CH1, thereby hindering and significantly reducing the formation of unwanted pairings such as C1-CH, C1-CL, C2-CH and C2-CL. Due to the specific association of C1-C2, VH1 specifically pairs with VL1 and thereby forms the first antigen binding site, and CH1 specifically pairs with CL, thereby allowing the specific pairing of VH2-VL2 that provides the second antigen binding site. Accordingly, the first antigen binding portion and the second antigen binding portion are less prone to mispairing, and compared to the case where the first and the second antigen binding portions are both corresponding portions of natural Fab (e.g., in the form of VH1-CH1, VL1-CL, VH2-CH1 and VL2-CL), the mispairing between, for example, VH1-VL2, VH2-VL1, VH1-VH2, and VL1-VL2 is significantly reduced.

[0522] In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complexes provided herein have significantly fewer mismatched products (e.g., a reduction of at least 1, 2, 3, 4, 5 or more mismatched products) and / or significantly higher yields (e.g., an increase of at least 10%, 20%, 30%, 40%, 50%, 60% or more in yield) when expressed from cells compared to a reference molecule expressed under comparable conditions, wherein the reference molecule is identical to the bispecific anti-CD3 / CD20 polypeptide complex except that C1 is replaced by native CH1 and C2 is replaced by native CL.

[0523] Antigen binding portion comprising engineered Cα and Cβ

[0524] The first antigen binding moiety provided by the present disclosure comprises a first antibody heavy chain variable domain operably connected to a first T cell receptor (TCR) constant region, and a first antibody light chain variable domain operably connected to a second TCR constant region, wherein the first TCR constant region and the second TCR constant region are associated via at least one non-natural interchain disulfide bond. The first antigen binding moiety comprises at least two polypeptide chains, each of which comprises a variable domain derived from an antibody and a constant region derived from TCR. Therefore, the first antigen binding moiety comprises a heavy chain variable domain and a light chain variable domain, which are respectively operably connected to a pair of TCR constant regions. In certain embodiments, the pairing of the TCR constant region in the first antigen binding moiety is an α / βTCR constant region. The TCR constant regions of the bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure can associate with each other to form a dimer through at least one non-natural disulfide bond.

[0525] Surprisingly, it was found that the first antigen binding portion provided by the present disclosure having at least one non-native disulfide bond can be recombinantly expressed and assembled into a desired conformation, which stabilizes the TCR constant region dimer and provides good antigen binding activity of the antibody variable region. In addition, it was found that the first antigen binding portion withstands conventional antibody engineering well, such as modification of glycosylation sites and removal of some native sequences. In addition, the bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure can be incorporated into a dual heterospecific form, which can be easily expressed and assembled with minimal or almost no mismatch of the antigen binding sequence due to the presence of the TCR constant region in the first antigen binding portion. Other advantages of the first antigen binding portion and construct provided by the present disclosure will be more apparent in the following disclosure.

[0526] In summary, the first antigen binding portion provided by the present disclosure comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody, which is operably linked to a first T cell receptor (TCR) constant region (C1), and the second polypeptide comprises, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody, which is operably linked to a second TCR constant region (C2), wherein: C1 and C2 can form a dimer, and a non-natural interchain disulfide bond between C1 and C2 can stabilize the dimer.

[0527] TCR constant region

[0528] The first antigen binding portion described in the present disclosure comprises an α or β constant region derived from a TCR.

[0529] The human TCR alpha chain constant region is called TRAC and its NCBI accession number is P01848.

[0530] There are two different variants of the human TCR beta chain constant region, referred to as TRBC1 and TRBC2 (IMGT nomenclature) (see also Toyonaga B et al., PNAs, Vol. 82, pp. 8624-8628, Immunology (1985)).

[0531] In the present disclosure, the first and second TCR constant regions of the first antigen binding moiety provided by the present disclosure are capable of forming a dimer, and the dimer comprises at least one non-native interchain disulfide bond between the TCR constant regions that is capable of stabilizing the dimer.

[0532] The term "dimer" used in the present disclosure refers to an associated structure formed by two molecules (such as polypeptides or proteins) via covalent or non-covalent interactions. Homodimers or homodimerizations are formed by two identical molecules, while heterodimers or heterodimerizations are formed by two different molecules. The dimer formed by the first and second TCR constant regions is a heterodimer.

[0533] A "mutated" amino acid residue refers to an amino acid residue that is substituted, inserted or added and is different from the corresponding native residue in the native TCR constant region to which it corresponds. For example, if the amino acid residue at a particular site in the wild-type TCR constant region is referred to as a "native" residue, then its corresponding mutated residue is any residue that is different from the native residue but is located at the same position on the TCR constant region. The mutated residue can be a different residue that replaces the native residue at the same position, or a different residue that is inserted before the native residue and thus occupies its original position.

[0534] In the bispecific anti-CD3 / CD20 polypeptide complexes provided herein, the first and / or the second TCR constant region has been engineered to include one or more mutant amino acid residues responsible for forming the non-native interchain disulfide bond. In order to introduce such mutant residues into the TCR constant region, the coding sequence of the TCR region can be manipulated, for example, to replace the codons encoding the native residues with the codons encoding the mutant residues, or to insert the codons encoding the mutant residues before the codons of the native residues.

[0535] In the bispecific anti-CD3 / CD20 polypeptide complexes provided herein, the first and / or the second TCR constant region has been engineered to contain one or more mutated cysteine ​​residues such that, upon replacement of the cysteine ​​residues, a non-native interchain disulfide bond can be formed between the two TCR constant regions.

[0536] The non-natural interchain disulfide bonds can stabilize the first antigen binding portion. This stabilization effect can be embodied in various ways. For example, the presence of mutant amino acid residues or non-natural interchain disulfide bonds can enable the bispecific anti-CD3 / CD20 polypeptide complex to be stably expressed, and / or expressed at a high level, and / or associated to a stable complex with an ideal biological activity (e.g., antigen binding activity), and / or expressed and assembled into a high level of an ideal stable complex with an ideal biological activity. The ability of the interchain disulfide bond to stabilize the first and second TCR constant regions can be assessed using suitable methods known in the art (such as displaying molecular weight on SDS-PAGE, or measuring thermal stability by differential scanning calorimetry (DSC) or differential scanning fluorescence (DSF)). In an illustrative example, if the product shows a molecular weight comparable to the molecular weight of the combination of the first and second polypeptides, it can be confirmed by SDS-PAGE that a stable first antigen binding portion provided by the present disclosure has been formed. In certain embodiments, the first antigen binding portion described in the present disclosure is stable, embodied in that its thermal stability is not less than 50%, 60%, 70%, 80% or 90% of that of a natural Fab. In certain embodiments, the first antigen binding moiety described in the present disclosure is stable, as reflected in that its thermal stability is comparable to that of natural Fab.

[0537] Without wishing to be bound by any theory, it is believed that the non-natural interchain disulfide bond formed between the first and second TCR constant regions in the first antigen binding moiety can stabilize the heterodimer of the TCR constant region, thereby enhancing the correct folding level, structural stability and / or expression level of the heterodimer and the first antigen binding moiety. Unlike the natural TCR anchored on the cell membrane on the surface of the T cell, the heterodimer of the natural TCR extracellular domain was found to be more unstable, although it is similar to the antibody Fab in 3D structure. In fact, the instability of the natural TCR in the dissolved state was a significant obstacle to its crystal structure being difficult to elucidate (see Wang, Protein Cell, 5 (9), pp.649-652 (2014)). By introducing a pair of cysteine ​​(Cys) mutations into the TCR constant region and thereby enabling the formation of non-natural interchain disulfide bonds, the first antigen binding moiety can be stably expressed while retaining the antigen binding ability of the antibody variable region.

[0538] The TCR constant region comprising a mutated residue is also referred to as an "engineered" TCR constant region in the present disclosure. In certain embodiments, the first TCR constant region (C1) of the bispecific anti-CD3 / CD20 polypeptide complex comprises an engineered TCR alpha chain (Cα), and the second TCR constant region (C2) comprises an engineered TCR beta chain (Cβ). In the bispecific anti-CD3 / CD20 polypeptide complex provided herein, C1 comprises an engineered Cβ, and C2 comprises an engineered Cα.

[0539] In the bispecific anti-CD3 / CD20 polypeptide complexes provided herein, the engineered TCR constant region comprises one or more mutated cysteine ​​residues, wherein the one or more mutated residues are contained within the contact interface of the first and / or the second engineered constant region.

[0540] The term "contact interface" as used in the present disclosure refers to a specific region on the polypeptide where the polypeptides interact / associate with each other. The contact interface comprises one or more amino acid residues that are capable of interacting with corresponding amino acid residues that are in contact or association when the interaction occurs. The amino acid residues in the contact interface may or may not be in a continuous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be separated from each other at different positions on the linear sequence.

[0541] In certain embodiments, one or more disulfide bonds may be formed between the engineered Ca and the engineered Cβ. In certain embodiments, the pairing of the cysteine ​​residues may form a non-native interchain disulfide bond.

[0542] "XnY" used throughout this disclosure with respect to a TCR constant region is intended to indicate that the nth amino acid residue X on the TCR constant region is substituted with amino acid residue Y, wherein X and Y are the single letter abbreviations of the specific amino acid residues, respectively.

[0543] In the bispecific anti-CD3 / CD20 polypeptide complex provided herein, the engineered Cβ comprises or is SEQ ID NO:61, and the engineered Ca comprises or is SEQ ID NO:62.

[0544] The sequences of SEQ ID NO:61 and SEQ ID NO:62 are provided below:

[0545] LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA(SEQ ID NO:61)

[0546] PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS(SEQ ID NO:62)

[0547] In the bispecific anti-CD3 / CD20 polypeptide complex provided in the present disclosure, in the first antigen binding portion provided in the present disclosure, one or more native glycosylation sites present in the native TCR constant region may be modified (e.g., removed). The term "glycosylation site" used in the present disclosure refers to an amino acid residue with a carbohydrate moiety (e.g., an oligosaccharide structure) attached to its side chain when used for a polypeptide sequence. Glycosylation of polypeptides (such as antibodies) is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue, such as an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of native glycosylation sites can be conveniently achieved by altering the amino acid sequence such that one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) or one or more of the serine or threonine residues (for O-linked glycosylation sites) present in the sequence are replaced.

[0548] In the first antigen binding portion provided in the present disclosure, at least one native glycosylation site is not present in the engineered TCR constant region (e.g., in the first and / or second TCR constant region). Without wishing to be bound by any theory, it is believed that the first antigen binding portion described in the present disclosure can allow the removal of all or part of the glycosylation sites without affecting protein expression and stability, which is contrary to existing teachings, i.e., the presence of N-linked glycosylation sites on TCR constant regions such as Cα (i.e., N34, N68, and N79) and Cβ (i.e., N69) is necessary for protein expression and stability (see Wu et al., Mabs, 7:2, 364-376, 2015).

[0549] In the first antigen binding portion provided by the present disclosure, the constant region derived from TCR is operably linked to the variable region derived from an antibody.

[0550] In certain embodiments, the first antibody variable domain (VH) is fused to the first TCR constant region (C1) at a first joining domain, and the first antibody variable domain (VL) is fused to the second TCR constant region (C2) at a second joining domain.

[0551] The term "joining domain" used in the present disclosure refers to the junction or border region where two amino acid sequences fuse or combine. In certain embodiments, the first joining domain comprises at least a portion of the C-terminal fragment of an antibody V / C joiner, and the second joining domain comprises at least a portion of the N-terminal fragment of a TCR V / C joiner.

[0552] The term "antibody V / C engager" used in the present disclosure refers to the boundary between an antibody variable domain and a constant domain, such as the boundary between a heavy chain variable domain and a CH1 domain or between a light chain variable domain and a light chain constant domain. Similarly, the term "TCR V / C engager" refers to the boundary between a TCR variable domain and a constant domain, such as the boundary between a TCR α variable domain and a constant domain or between a TCR β variable domain and a constant domain.

[0553] In certain embodiments, the first polypeptide comprises a sequence comprising a domain operably linked as in formula (I): VH-HCJ-C1, and the second polypeptide comprises a sequence comprising a domain operably linked as in formula (II): VL-LCJ-C2, wherein:

[0554] VH is the heavy chain variable domain of the antibody; HCJ is the first joining domain as defined above; C1 is the first TCR constant domain as defined above; VL is the light chain variable domain of the antibody; LCJ is the second joining domain as defined above; C2 is the second TCR constant domain as defined above.

[0555] Antibody variable region

[0556] The bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure comprises a first antigen binding portion associated with a second antigen binding portion, and one of them specifically binds to CD3 and the other specifically binds to CD20. In the bispecific anti-CD3 / CD20 polypeptide complex provided by the present disclosure, the first antigen binding portion comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1) of a first antibody, and the second antigen binding portion comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody, wherein the first antibody and the second antibody are different and are selected from the group consisting of anti-CD3 antibodies and anti-CD20 antibodies. In certain embodiments, the first antibody is an anti-CD3 antibody and the second antibody is an anti-CD20 antibody. In certain other embodiments, the first antibody is an anti-CD20 antibody and the second antibody is an anti-CD3 antibody.

[0557] In a conventional native antibody, the variable region comprises three CDR regions separated by flanking framework (FR) regions, for example as listed from N-terminus to C-terminus by the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0558] a) Anti-CD3 binding moiety

[0559] In the bispecific anti-CD3 / CD20 polypeptide complexes provided herein, the first antigen binding moiety or the second antigen binding moiety is an anti-CD3 binding moiety.

[0560] In certain embodiments, the anti-CD3 binding portion is derived from antibodies W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP shown in Table A below. The CDR sequences of the anti-CD3 binding portions of the W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP antibodies are provided below.

[0561] Table A

[0562]

[0563] The heavy chain and kappa light chain variable region sequences of the anti-CD3 binding portions of the W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP antibodies are provided below.

[0564] VH - amino acid sequence (SEQ ID NO: 25):

[0565] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLEWMGWIFPGNDNIKYSEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIDSVSIYYFDYWGQGTLVTVSS

[0566] VH - Nucleic acid sequence (SEQ ID NO:53):

[0567] caggtgcaactcgtgcagtctggagctgaagtgaagaagcctgggtcttcagtcaaggtcagttgcaaggccagtgggtattccttcactacctactacatccactgggtgcggcaggcaccaggacaggggcttgagtggatgggctggatctttcccggcaacgataatattaagtacagcgagaagttcaaagggagggtcaccattaccgccgacaaatccacttccacagcctacatggagttgagcagcctgagatccgaggatacagccgtgtactactgtgccattgacagcgtgtccatctactactttgactactggggccagggcacactggtcacagtgagcagc

[0568] Vκ - Amino acid sequence (SEQ ID NO:26):

[0569] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGGGTKVEIK

[0570] Vκ - Nucleic acid sequence (SEQ ID NO:54):

[0571] gacatcgtcatgacccagtccccagactctttggcagtgtctctcggggaaagagctaccatcaactgcaagagcagccagtcccttctgaacagcaggaccaggaagaattacctcgcctggtaccaacagaagcccggacagcctcctaagctcctgatctactgg gcctcaacccggaagagtggagtgcccgatcgctttagcggggagcggctccgggacagatttcacactgacaatttcctccctgcaggccgaggacgtcgccgtgtattactgtactcagagcttcattctgcggacatttggcggcgggactaaagtggagattaag

[0572] In some embodiments, the heavy chain variable region sequence of the anti-CD3 binding portion of the W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP antibodies is an amino acid sequence variant of SEQ ID NO: 25, for example, an amino acid sequence in which amino acid SS is deleted at the C-terminus of SEQ ID NO: 25.

[0573] In certain embodiments, the anti-CD3 binding portion is derived from the antibodies W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP shown in Table B below. The CDR sequences of the anti-CD3 binding portions of the W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP antibodies are provided below.

[0574] Table B

[0575]

[0576] The heavy chain and kappa light chain variable region sequences of the anti-CD3 binding portions of the W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP antibodies are provided below.

[0577] VH - amino acid sequence (SEQ ID NO: 27):

[0578] QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWGQGTLVTVSS

[0579] VH - nucleic acid sequence (SEQ ID NO:55):

[0580] caggtgcagcttgtgcagtctggggcagaagtgaagaagcctgggtctagtgtcaaggtgtcatgcaaggctagcgggttcgcctttactgactactacatccactgggtgcggcaggctcccggacaagggttggagtggatgggatggatctccccaggcaatgtcaacacaaagtacaacgagaacttcaaaggccgcgtcaccattaccgccgacaagagcacctccacagcctacatggagctgtccagcctcagaagcgaggacactgccgtctactactgtgccagggatgggtactccctgtattactttgattactggggccagggcacactggtgacagtgagctcc

[0581] Vκ - amino acid sequence (SEQ ID NO:28): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRQSGVPDRFSGS

[0582] GSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVEIK

[0583] Vκ - nucleic acid sequence (SEQ ID NO:56):

[0584] gatatcgtgatgaccccagagcccagactcccttgctgtctccctcggcgaaagagcaaccatcaactgcaagagctcccaaagcctgctgaactccaggaccaggaagaattacctggcctggtatcagcagaagcccggccagcctcctaagctgctcatctactgg gcctccacccggcagtctggggtgcccgatcggtttagtggatctggggagcgggacagacttcacattgacaattagctcactgcaggccgaggacgtggccgtctactactgtactcagagccacactctccgcacattcggcggagggactaaagtggagattaag

[0585] In some embodiments, the heavy chain variable region sequence of the anti-CD3 binding portion of the W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP antibodies is an amino acid sequence variant of SEQ ID NO: 27, for example, an amino acid sequence lacking amino acid SS at the C-terminus of SEQ ID NO: 27.

[0586] The anti-CD3 binding portions provided herein also comprise suitable framework region (FR) sequences, so long as the anti-CD3 binding portions can specifically bind to CD3.

[0587] The anti-CD3 antibodies of the present disclosure are effective against cells expressing CD3 (e.g., CD4 + T cells) and can activate human T cells and trigger the release of cytokines such as TNFα and IFNγ.

[0588] The binding affinity of the anti-CD3 binding moieties provided by the present disclosure can be determined by K D The value is expressed as, which represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on Antigen binding affinity (e.g., K) can be appropriately determined using appropriate methods known in the art, including, for example, flow cytometry assays. DIn some embodiments, the binding of antibodies to antigens at different concentrations can be determined by flow cytometry, and the determined mean fluorescence intensity (MFI) can first be plotted against the antibody concentration, and then the dependence of the specific binding fluorescence intensity (Y) on the antibody concentration (X) can be fitted to the single-site saturation equation using Prism version 5 (GraphPad Software, San Diego, California): Y = B max *X / (K D +X), and thus calculate K D Value, where B max It refers to the maximum specific binding of the antibody to be tested to the antigen.

[0589] In certain embodiments, the anti-CD3 binding moiety provided by the present disclosure can specifically bind to human CD3 or recombinant human CD3 expressed on the cell surface. CD3 is a receptor expressed on cells. Recombinant CD3 is a soluble CD3 that is recombinantly expressed and not associated with a cell membrane. Recombinant CD3 can be prepared by a variety of recombinant techniques. In one example, a CD3 DNA sequence encoding a human CD3 extracellular domain (NP_000724.1) (Met1-Asp126) can be fused to a polyhistidine tag at the C-terminus in an expression vector, and then transfected and expressed in 293E cells, and purified by nickel affinity chromatography.

[0590] In some embodiments, the present disclosure provides anti-CD3 binding moieties capable of binding at a rate of no more than 5×10 -9 M, not more than 4×10 -9 M, not more than 3×10 -9 M, not more than 2×10 -9 M. No more than 10 -9 M, not more than 5×10 -10 M, not more than 4×10 -10 M, not more than 3×10 -10 M, not more than 2×10 -10 M. No more than 10 -10 M, not more than 5×10 -11 M or not more than 4×10 - 11 M, not more than 3×10 -11 M or not more than 2×10 -11 M or not more than 10 -11 The binding affinity (K D ) specifically binds to human CD3 expressed on the cell surface, the K D Values ​​were determined by flow cytometry.

[0591] In certain embodiments, the anti-CD3 binding portions provided by the present disclosure cross-react with cynomolgus CD3 (e.g., cynomolgus CD3 expressed on the surface of a cell, or soluble recombinant cynomolgus CD3).

[0592] The binding of the anti-CD3 binding portion to recombinant CD3 or CD3 expressed on the cell surface can be determined by methods known in the art, such as sandwich methods (such as ELISA), Western blot, flow cytometry and other binding assays. In certain embodiments, the anti-CD3 binding portion provided by the present disclosure has an EC of no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.06 nM, no more than 0.07 nM or no more than 0.08 nM. 50 (i.e. 50% binding concentration) specifically binds to recombinant human CD3. 50 In certain embodiments, the anti-CD3 binding moieties provided herein have an EC of no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM, no more than 1 nM, no more than 2 nM, no more than 3 nM, no more than 4 nM, no more than 5 nM, no more than 6 nM, no more than 7 nM, no more than 8 nM, no more than 9 nM, or no more than 10 nM. 50 Specifically binds to human CD3 expressed on the cell surface. 50 Values ​​were determined by flow cytometry.

[0593] In certain embodiments, the anti-CD3 binding portion binds to cynomolgus monkey CD3 with a similar binding affinity as human CD3.

[0594] In certain embodiments, the anti-CD3 binding moieties provided herein have an EC no greater than 0.001 nM, no greater than 0.005 nM, no greater than 0.01 nM, no greater than 0.02 nM, no greater than 0.03 nM, no greater than 0.04 nM, or no greater than 0.05 nM. 50 Specifically binds to recombinant cynomolgus monkey CD3. 50 The values ​​were determined by ELISA.

[0595] In certain embodiments, the anti-CD3 binding moieties provided by the present disclosure have specific binding affinity to human CD3 sufficient for diagnostic and / or therapeutic applications.Many therapeutic regimens modulate T cell immunity by targeting TCR signaling, particularly through clinically used anti-human CD3 monoclonal antibodies.

[0596] b) Anti-CD20 antibody

[0597] In the bispecific anti-CD3 / CD20 polypeptide complexes provided herein, the first antigen binding moiety or the second antigen binding moiety is an anti-CD20 binding moiety.

[0598] In certain embodiments, the anti-CD20 binding portion is derived from antibodies W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP shown in Table A' below. The CDR sequences of the anti-CD20 binding portions of the W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP antibodies are provided below.

[0599] Table A'

[0600]

[0601] The heavy chain and kappa light chain variable region sequences of the anti-CD20 binding portions of the W3278-T2U3.E17R-1.uIgG4.SP and W3278-U3T2.F18R-1.uIgG4.SP antibodies are provided below.

[0602] VH - amino acid sequence (SEQ ID NO: 29):

[0603] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS

[0604] VH-nucleic acid sequence (SEQ ID NO:57):

[0605] gaggtgcaattggtggagagcggaggagggctcgtgcagcctggaagatctcttaggctgagttgcgctgcatctgggttcacattcaacgactacgccatgcactgggtgaggcaggctcccggcaaagggctggaatgggtgtcaactatctcctggaactccggcagcatcggctacgccgatagcgtcaagggccggtttacaatttcccgcgataacgccaagaagtccctgtacctgcagatgaacagcctgcgggccgaggatactgccctctactactgtgccaaggacattcagtacgggaattactattacgggatggacgtctggggccaggggaccaccgtgacagtcagctcc

[0606] Vκ - amino acid sequence (SEQ ID NO:30):

[0607] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK

[0608] Vκ - nucleic acid sequence (SEQ ID NO:58):

[0609] gaaatcgtgctgacccagtccccagcaaccctctccctttctcctggagagagagctaccctcagctgtagggcctcacagtctgtctccagttacctggcttggtaccagcagaaacccgggcaggcccctaggttgctgatctacgacgccagcaatagggccactggcatcccagcccggttttccggaagcggcagcgggacagatttcacactcactattagcagcctggagcccgaggacttcgccgtgtactattgccagcagcggtccaactggcccattacatttggccaagggacacgcctggagattaag

[0610] In certain embodiments, the anti-CD20 binding portion is derived from the antibodies W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP shown in Table B' below. The CDR sequences of the anti-CD20 binding portions of the W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP antibodies are provided below.

[0611] Table B'

[0612]

[0613] The heavy chain and kappa light chain variable region sequences of the anti-CD20 binding portions of the W3278-T3U2.F16-1.uIgG4.SP, W3278-U2T3.F18R-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP antibodies are provided below.

[0614] VH - amino acid sequence (SEQ ID NO:31):

[0615] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA

[0616] VH-nucleic acid sequence (SEQ ID NO:59):

[0617] caggtccagctgcagcagcccggagccgaactggtcaaacccggggctagcgtgaaaatgtcttgcaaagcaagtggttacacattcacttcctataacatgcactgggtgaagcagacacctgggcgaggtctggaatggatcggcgccatctacccaggcaacggagacactagctataatcagaagtttaaaggaaaggccaccctgacagctgataagtccagctctaccgcttacatgcagctgagttcactgacaagtgaggactcagcagtgtactattgcgcccgttctacctactatggcggagattggtatttcaatgtgtggggcgccggtaccacagtcaccgtgtccgcc

[0618] Vκ - Amino acid sequence (SEQ ID NO:32):

[0619] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0620] Vκ - Nucleic acid sequence (SEQ ID NO:60):

[0621] cagattgtcctgagccagagccctgccatcctgtctgctagtcccggcgagaaggtgaccatgacatgcagggcatccagctctgtctcctacatccactggttccagcagaagcccgggagttcacctaaaccatggatctacgctacatccaacctggcaagcggtgtgcctgtcaggttttcaggttccggcagcggaacatcttacagtctgactatttctcgggtggaggccgaagacgccgctacctactattgccagcagtggacctccaatccccctacattcggcggagggactaagctggagatcaaa

[0622] The anti-CD20 binding portions provided herein also comprise suitable framework region (FR) sequences, so long as the anti-CD20 binding portions can specifically bind to CD20.

[0623] In some embodiments, the anti-CD20 binding moieties provided by the present disclosure can be present at a rate of no more than 5×10 -9 M, not more than 1×10 -9 M, not more than 9×10 -10 M, not more than 8×10 -10 M, not more than 7×10 -10 M, not more than 6×10 -10 M, not more than 5×10 -10 M, not more than 4×10 -10 M, not more than 3×10 -10 M, not more than 2×10 -10 M or not more than 1×10 -10 The binding affinity (K D ), specifically binds to human CD20 expressed on the cell surface, the K D Values ​​were determined by flow cytometry.

[0624] In certain embodiments, the anti-CD20 binding portions provided herein cross-react with cynomolgus CD20 (e.g., cynomolgus CD20 expressed on the surface of a cell or soluble recombinant cynomolgus CD20).

[0625] The binding of the anti-CD20 binding portion to CD20 expressed on cells can be determined by methods known in the art, such as sandwich methods (such as ELISA), Western blot, flow cytometry and other binding assays. In certain embodiments, the anti-CD20 binding portion provided by the present disclosure has an EC of no more than 0.01 nM, no more than 0.02 nM, no more than 0.03 nM, no more than 0.04 nM, no more than 0.05 nM, no more than 0.1 nM, no more than 0.2 nM, no more than 0.3 nM, no more than 0.4 nM, no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM or no more than 1 nM. 50 Specifically binds to human CD20 expressed on cells. 50 Values ​​were determined by flow cytometry.

[0626] In certain embodiments, the anti-CD20 binding moieties bind to cynomolgus CD20 with a binding affinity similar to that of human CD20. In certain embodiments, the anti-CD20 binding moieties provided herein bind to cynomolgus CD20 with an EC no greater than 0.2 nM, no greater than 0.5 nM, no greater than 0.8 nM, no greater than 1 nM, no greater than 2 nM, or no greater than 3 nM.50 Specifically binds to cynomolgus monkey CD20 expressed on cells. 50 Values ​​were determined by flow cytometry.

[0627] In certain embodiments, the anti-CD20 binding portions described herein have an EC of no more than 1 pM, no more than 2 pM, no more than 3 pM, no more than 4 pM, no more than 5 pM, no more than 6 pM, no more than 7 pM, no more than 8 pM, no more than 9 pM, no more than 10 pM, no more than 11 pM, no more than 12 pM, no more than 13 pM, no more than 14 pM, no more than 15 pM, no more than 16 pM, no more than 17 pM, no more than 18 pM, no more than 19 pM, no more than 20 pM, no more than 21 pM, no more than 22 pM, no more than 23 pM, no more than 24 pM, no more than 25 pM, no more than 30 pM, no more than 35 pM, no more than 40 pM, no more than 45 pM, or no more than 50 pM. 50 Internalized by CD20-expressing cells, the EC 50 Values ​​were determined by Fab-Zap assay.

[0628] Bispecific anti-CD3 / CD20 peptide complex

[0629] In certain embodiments, the first and / or the second antigen binding moiety is multivalent, such as bivalent, trivalent, tetravalent. The term "valence" used in the present disclosure refers to the presence of a specified number of antigen binding sites in a given molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" respectively indicate that there are two binding sites, four binding sites and six binding sites in the antigen binding molecule. If both binding sites are used to specifically bind to the same antigen or the same epitope, the bivalent molecule can be monospecific. Similarly, a trivalent molecule can be bispecific, for example when two binding sites are monospecific for the first antigen (or epitope), and the third binding site is specific for the second antigen (or epitope). In certain embodiments, the first and / or the second antigen binding moiety in the bispecific anti-CD3 / CD20 polypeptide complex described in the present disclosure can be bivalent, trivalent or tetravalent, having at least two binding sites for the same antigen or epitope. In certain embodiments, this provides a stronger binding to the antigen or epitope than a monovalent corresponding antibody. In certain embodiments, in a bivalent antigen binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (ie, have the same sequence) or structurally different (ie, have different sequences but the same specificity).

[0630] In certain embodiments, the first and / or the second antigen binding moiety is multivalent and comprises two or more antigen binding sites operably associated with each other (with or without a spacer).

[0631] In certain embodiments, the second antigen binding moiety comprises two or more Fabs of the second antibody. The two Fabs can be operably associated with each other, for example, the first Fab can be covalently attached to the second Fab via the heavy chain, with or without a spacer.

[0632] In certain embodiments, the first antigen binding moiety is linked to a first dimerization domain, and the second antigen binding moiety is linked to a second dimerization domain. The term "dimerization domain" as used in the present disclosure refers to a peptide domain that is capable of associating with each other to form a dimer, or in some examples, a peptide domain that enables two peptides to spontaneously dimerize.

[0633] In certain embodiments, the first dimerization domain may be associated with the second dimerization domain. The association may be achieved via any suitable interaction or connection or bonding (e.g., via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof). Exemplary dimerization domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody CH3 domains, and other suitable protein monomers capable of dimerization and mutual association. Hinge regions, CH2 and / or CH3 domains may be derived from any antibody isotype, such as IgG1, IgG2, and IgG4.

[0634] "Disulfide bond" refers to a covalent bond having a RSS-R' structure. The amino acid cysteine ​​contains a sulfhydryl group, which can form a disulfide bond with a second sulfhydryl group, for example, from another cysteine ​​residue. The disulfide bond can be formed between the sulfhydryl groups of two cysteine ​​residues, respectively present on two polypeptide chains, thereby forming an interchain bridge or interchain bond.

[0635] A hydrogen bond is formed by an electrostatic interaction between two polar groups when a hydrogen atom covalently binds to a highly electronegative atom such as nitrogen, oxygen or fluorine. A hydrogen bond can be formed between a backbone oxygen (e.g., a sulfide group) and an amide hydrogen (a nitrogen group) of each of two residues in a polypeptide, such as the nitrogen group in Asn with the oxygen group in His, or the oxygen group in Asn with the nitrogen group in Lys. Hydrogen bonds are stronger than van der Waals interactions but weaker than covalent or ionic bonds and are critical for maintaining secondary and tertiary structure. For example, an alpha helix is ​​formed when the spacing of amino acid residues occurs regularly between positions i and i+4, while a beta sheet is a peptide segment of 3-10 amino acids in length that forms when two peptide segments are connected by at least two or three backbone hydrogen bonds, forming a twisted, wrinkled sheet.

[0636] Electrostatic interactions are non-covalent interactions that are important in protein folding, stability, flexibility and function, and include ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions can be formed in polypeptides, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp on the first chain and Arg, Val or Thr on the second chain.

[0637] Salt bridges are short-range electrostatic interactions that occur primarily between anionic carboxylates from Asp or Glu and cationic ammonium from Lys or guanidine from Arg. Salt bridges are pairs of oppositely charged residues that are spatially close in the native protein structure. Charged and polar residues in a predominantly hydrophobic interface can serve as hotspots for binding. Other residues, including residues with ionizable side chains such as His, Tyr, and Ser, can also participate in the formation of salt bridges.

[0638] Hydrophobic interactions can be formed between one or more of Val, Tyr, and Ala on the first strand and one or more of Val, Leu, and Trp on the second strand, or between His and Ala on the first strand and Thr and Phe on the second strand (see Brinkmann et al., 2017, supra).

[0639] In certain embodiments, the first and / or the second dimerization domain comprises at least a portion of an antibody hinge region. In certain embodiments, the first and / or the second dimerization domain may further comprise an antibody CH2 domain and / or an antibody CH3 domain. In certain embodiments, the first and / or the second dimerization domain comprises at least a portion of a hinge-Fc region, i.e., a hinge-CH2-CH3 domain. In certain embodiments, the first dimerization domain may be operably connected to the C-terminus of the first TCR constant region. In certain embodiments, the second dimerization domain may be operably connected to the C-terminus of the antibody CH1 constant region of the second antigen binding moiety.

[0640] In the bispecific anti-CD3 / CD20 polypeptide complex provided herein, the first dimerization domain is operably connected to the C-terminus of the engineered TCR constant region and together form a chimeric constant region. That is, the chimeric constant region includes the first dimerization domain operably connected to the engineered TCR constant region.

[0641] In certain embodiments, the chimeric constant region comprises an engineered Cβ attached to a first hinge-Fc region derived from IgG1, IgG2, or IgG4.

[0642] In certain embodiments, the chimeric constant region further comprises a first antibody CH2 domain and / or a first antibody CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain attached to the C-terminus of the third engagement domain.

[0643] These chimeric constant regions and second TCR constant region pairs are useful because they can be manipulated to fuse to the desired antibody variable region to provide the bispecific anti-CD3 / CD20 polypeptide complex described in the present disclosure. For example, the antibody heavy chain variable region can be fused to the chimeric constant region (including C1) to provide the first polypeptide chain of the bispecific anti-CD3 / CD20 polypeptide complex described in the present disclosure; and similarly, the antibody light chain variable region can be fused to the second TCR constant region (including C2) to provide the second polypeptide chain of the bispecific anti-CD3 / CD20 polypeptide complex described in the present disclosure.

[0644] In certain embodiments, the second dimerization domain includes a hinge region. The hinge region may be derived from an antibody, such as IgG1, IgG2 or IgG4. In certain embodiments, the second dimerization domain may optionally further include an antibody CH2 domain and / or an antibody CH3 domain, such as a hinge-Fc region. The hinge region may be attached to an antibody heavy chain (e.g., Fab) of the second antigen binding site.

[0645] In the bispecific anti-CD3 / CD20 polypeptide complex, the first and second dimerization domains are capable of associating into dimers. In certain embodiments, the first and second dimerization domains are different and are associated in a manner that is not conducive to homodimerization and / or is conducive to heterodimerization. For example, the first and second dimerization domains can be selected so that they are different and they preferentially form heterodimers with each other rather than homodimers with themselves. In certain embodiments, the first and second dimerization domains can associate into heterodimers via the formation of knobs-into-holes, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility, and the “knobs-into-holes” are described in Ridgway et al., Protein Engineering, 9 (7), pp. 617–21 (1996); Merchant et al., Nature Biotechnology, 16 (7), pp. 677–681 (1998), etc.

[0646] In certain embodiments, the first and second dimerization domains comprise a CH2 and / or CH3 domain, each mutated to form a handle-into-hole. The handle can be obtained by replacing a small amino acid residue with a large amino acid residue in the first CH2 / CH3 polypeptide, and the hole can be obtained by replacing a large amino acid residue with a small amino acid residue. For details on the mutation sites of the handle-into-hole, see Ridgway et al., 1996, supra, Spiess et al., 2015, supra, and Brinkmann et al., 2017, supra.

[0647] Bispecific formats

[0648] In the bispecific anti-CD3 / CD20 polypeptide complex described herein, the first antigen binding moiety and the second binding moiety can be associated to form an Ig-like structure. The Ig-like structure is similar to a natural antibody, has a Y-shaped structure, has two arms for antigen binding and a stem for association and stability. The similarity to natural antibodies can provide a variety of advantages, such as good in vivo pharmacokinetics, ideal immune response and stability. It has been found that the Ig-like structure comprising the first antigen binding moiety provided by the present disclosure and the second antigen binding moiety provided by the present disclosure has a thermal stability comparable to that of Ig (e.g., IgG). In certain embodiments, the Ig-like structure provided by the present disclosure is at least 70%, 80%, 90%, 95% or 100% of natural IgG.

[0649] In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises four polypeptide chains: i) VH1-C1-hinge-CH2-CH3; ii) VL1-C2; iii) VH2-CH1-hinge-CH2-CH3, and iv) VL2-CL, wherein C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain bond, and the two hinge regions and / or the two CH3 domains are capable of forming one or more interchain bonds that can assist dimerization.

[0650] When compared to other forms of bispecific polypeptide complexes, the bispecific anti-CD3 / CD20 polypeptide complexes provided by the present disclosure have a longer in vivo half-life and are relatively easy to manufacture.

[0651] Bispecific anti-CD3 / CD20 peptide complex sequence

[0652] In some embodiments, the first antigen binding portion of the bispecific anti-CD3 / CD20 polypeptide complex is capable of specifically binding to CD3, and the second antigen binding portion is capable of specifically binding to CD20. In other embodiments, the first antigen binding portion of the bispecific anti-CD3 / CD20 polypeptide complex is capable of specifically binding to CD20, and the second antigen binding portion is capable of specifically binding to CD3.

[0653] In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 (W3278-T2U3.E17R-1.uIgG4.SP antibody), as described in Example 2. In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 (W3278-T3U2.F16-1.uIgG4.SP antibody), as described in Example 2. In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, as described in Example 2. In a certain embodiment, the bispecific anti-CD3 / CD20 polypeptide complex comprises five polypeptide chains: a) a first polypeptide chain having a sequence as shown in SEQ ID NO: 41; b) a second polypeptide chain having a sequence as shown in SEQ ID NO: 42; c) a third polypeptide chain having a sequence as shown in SEQ ID NO: 43; d) a fourth polypeptide chain having a sequence as shown in SEQ ID NO: 43; and e) a fifth polypeptide chain having a sequence as shown in SEQ ID NO: 44. For example, in a specific embodiment, the bispecific anti-CD3 / CD20 polypeptide complex is a W3278-U2T3.F18R-1.uIgG4.SP antibody, which comprises two anti-CD20 binding moieties, one of which has a heavy chain VH-CH1 domain operably linked to a heavy chain VH domain of an anti-CD3 binding moiety, as shown in Example 2. In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 (W3278-U3T2.F18R-1.uIgG4.SP antibody), as described in Example 2. In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 (W3278-T3U2.F17R-1.uIgG4.SP antibody), as described in Example 2. In such embodiments, the first antigen binding moiety binds to CD3 and the second antigen binding moiety binds to CD20.

[0654] In certain embodiments, the bispecific anti-CD3 / CD20 polypeptide complex comprises four polypeptide chains, comprising: i) VH1 operably linked to a first chimeric constant region; ii) VL1 operably linked to a second chimeric constant region; iii) VH2 operably linked to a conventional antibody heavy chain constant region, and iv) VL2 operably linked to a conventional antibody light chain constant region. In certain embodiments, the first chimeric constant region may comprise C1-hinge-CH2-CH3, each portion of which is as defined above. In certain embodiments, the second chimeric constant region may comprise C2, which is as defined above. In certain embodiments, the conventional antibody heavy chain constant region may comprise CH1-hinge-CH2-CH3, each portion of which is as defined above. In certain embodiments, the conventional antibody light chain constant region may comprise CL, which is as defined above.

[0655] In certain embodiments of the present disclosure, one or more amino acids in the natural glycosylation sites at positions 193, 182, 203, 206, and 207 of the polypeptide chain shown in SEQ ID NO: 44 are modified, preferably the amino acid at position 193 is modified. In certain embodiments of the present disclosure, the modification includes one or more of S193X, S182X, S203X, S206X, and S207X, wherein X is any amino acid other than serine (Ser) and / or threonine (Thr). In certain preferred embodiments of the present disclosure, the modification is S193X, wherein X is alanine (Ala), glycine (Gly), proline (Pro), or valine (Val). In certain embodiments of the present disclosure, the mutation eliminates the O-glycosylation site, and the O-glycosylation type is an O-sugar of Core1 configuration, and the structural formula is NeuAc-Gal-GalNAc or NeuAc-Gal-(NeuAc)GalNAc.

[0656] As described above, in the bispecific anti-CD3 / CD20 polypeptide complex of the present invention, the natural glycosylation sites in the corresponding polypeptide chains are modified. Compared with the unmodified bispecific anti-CD3 / CD20 polypeptide complex, the obtained bispecific anti-CD3 / CD20 polypeptide complex is closer to the natural antibody, has significantly reduced immunogenicity, increased half-life, and improved drugability.

[0657] Preparation method

[0658] The present disclosure provides isolated nucleic acids or polynucleotides encoding the bispecific anti-CD3 x CD20 polypeptide complexes of the present disclosure.

[0659] The term "nucleic acid" or "polynucleotide" used in the present disclosure refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double-stranded form. Unless explicitly limited, the term encompasses polynucleotides containing known natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific polynucleotide sequence also implies conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, homologous genes, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0660] Recombinant techniques can be used to construct nucleic acids or polynucleotides encoding the bispecific anti-CD3 x CD20 polypeptide complex of the present disclosure. To this end, DNA encoding the antigen binding portion (such as CDR or variable region) of the parent antibody can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). DNA encoding the TCR constant region can also be obtained in the same manner. As an example, a polynucleotide sequence encoding the variable domain (VH) and a polynucleotide sequence encoding the first TCR constant region (C1) are obtained and operably connected to allow transcription and expression in a host cell to produce the first polypeptide. Similarly, a polynucleotide sequence encoding VL is operably connected to a polynucleotide sequence encoding C1 so as to allow expression of the second polynucleotide in a host cell. If desired, the encoding polynucleotide sequence of one or more spacers can also be operably connected to other encoding sequences to allow expression of the desired product.

[0661] The encoding polynucleotide sequence may further be operably linked to one or more regulatory sequences, optionally in an expression vector, so that the expression or production of the first and the second polypeptide is feasible and under appropriate control.

[0662] Using recombinant techniques known in the art, the encoding polynucleotide sequence can be inserted into a vector for further cloning (amplification of the DNA) or for expression. In another embodiment, the polypeptide complex and bispecific anti-CD3 x CD20 polypeptide complex disclosed herein can be produced by homologous recombination known in the art. A variety of vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0663] The vectors containing the polynucleotide sequences described in the present disclosure can be introduced into host cells for cloning or gene expression. The term "host cell" used in the present disclosure refers to a cell into which an exogenous polynucleotide and / or vector is introduced. The host cells are transformed with the above-mentioned expression or cloning vectors and cultured in a culture medium.

[0664] In one aspect, the present disclosure provides a method for expressing the bispecific anti-CD3 x CD20 polypeptide complex of the present disclosure, comprising culturing the host cell of the present disclosure under conditions where the bispecific anti-CD3 x CD20 polypeptide complex is expressed.

[0665] In certain embodiments, the present disclosure provides a method for producing the bispecific anti-CD3 x CD20 polypeptide complex of the present disclosure, comprising a) introducing one or more polynucleotides encoding a first antigen binding moiety, including a first polynucleotide encoding a first polypeptide (the first polypeptide comprises a first heavy chain variable domain (VH) of a first antibody from N-terminus to C-terminus, which is operably linked to a first T cell receptor (TCR) constant region (C1)), a second polynucleotide encoding a second polypeptide (the second polypeptide comprises a first light chain variable domain (VL) of the first antibody from N-terminus to C-terminus, which is operably linked to a second TCR constant region (C2)) and one or more additional polynucleotides encoding a second antigen binding moiety into a host cell, wherein: C1 and C2 are capable of forming a dimer, and the non-natural interchain bond is capable of stabilizing the dimer of C1 and C2, the first antigen binding moiety and the second antigen binding moiety have reduced mismatching compared to the case where both the first antigen binding moiety and the second antigen binding moiety are corresponding parts of natural Fab, and the first antibody has a first antigen specificity and the second antibody has a second antigen specificity, and b) causing the host cell to express the bispecific anti-CD3 x CD20 polypeptide complex. CD20 polypeptide complex. In certain embodiments, the method further comprises isolating the bispecific anti-CD3 x CD20 polypeptide complex.

[0666] The bispecific anti-CD3 x CD20 polypeptide complex of the present disclosure prepared from the host cells can be purified by methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out, and affinity chromatography, wherein affinity chromatography is a preferred purification technique.

[0667] When the bispecific anti-CD3 x CD20 polypeptide complexes of the present disclosure include immunoglobulin Fc domains, protein A may be used as an affinity ligand, depending on the type and isotype of the Fc domain present in the polypeptide complex. Protein A can be used for purification of polypeptide complexes based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is suitable for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). Agarose is the most commonly used matrix for attachment of affinity ligands, but other matrices may also be used. Mechanically stable matrices such as controlled pore glass or poly(styrene)benzene can achieve faster flow rates and shorter processing times than with agarose.

[0668] When the bispecific anti-CD3 x CD20 polypeptide complex of the present disclosure comprises a CH3 domain, it can be purified using Bakerbond ABX.TM resin (JT Baker, Phillipsburg, New Jersey). Other protein purification techniques may also be used, such as fractionation in an ion exchange column, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (e.g., polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody to be recovered.

[0669] Following any preliminary purification steps, the mixture containing the polypeptide complex of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25 M salt concentration).

[0670] In certain embodiments, the bispecific anti-CD3 xCD20 polypeptide complexes of the present disclosure can be easily purified with high yield using conventional methods. One advantage of the bispecific anti-CD3 xCD20 polypeptide complexes is that the mismatch between the heavy chain and light chain variable domain sequences is significantly reduced. This reduces the generation of unwanted by-products and makes it possible to obtain a high purity product with high yield using a relatively simple purification process.

[0671] The following examples are provided to better illustrate the present disclosure, and they should not be construed as limiting the scope of the present disclosure. All of the following specific compositions, materials and methods, in whole or in part, are within the scope of the present disclosure. These specific compositions, materials and methods are not intended to limit the present disclosure, but are only intended to illustrate specific embodiments that fall within the scope of the present disclosure. Those skilled in the art can develop equivalent compositions, materials and methods without exerting creativity and without departing from the scope of the present disclosure. It should be understood that the various modifications made to the method of the present disclosure may still be included within the scope of the present disclosure. The inventor intends to include such changes within the scope of the present disclosure.

[0672] Example

[0673] Example 1

[0674] Material preparation and reference antibodies

[0675] 1. Material Preparation

[0676] Information on commercially available materials used in the examples is provided in Table 1.

[0677] Table 1. Commercially available materials

[0678]

[0679]

[0680] 2. Generate benchmark antibodies

[0681] Two benchmark antibodies, W327-BMK1 and W327-BMK4, were used as reference antibodies in the Examples.

[0682] The anti-human CD20 reference antibody BMK1 (rituximab) was generated based on the sequence of clone C2B8 from US patent application US20140004037 A1. The anti-CD3×CD20 reference bispecific antibody BMK4 (REGN1979) gene was synthesized according to the sequence in US patent application US20150266966 A1. The BMK antibody was expressed by Expi293 cells and then purified using protein A chromatography.

[0683] Example 2

[0684] Preparation of bispecific antibodies of the present disclosure

[0685] 1. Design and engineering of antibody and TCR chimeric proteins

[0686] TCR sequence

[0687] TCR is a heterodimeric protein composed of two chains. About 95% of human T cells have TCRs composed of α and β chains. Considering that more crystal structures are available for the β chain TRBC1, the TRBC1 sequence was selected as the main framework for designing the polypeptide complex ("WuXiBody") disclosed in the present disclosure. The typical amino acid sequence of TRBC1 can be found in Protein Data Bank (PDB) structure 4L4T.

[0688] Interchain disulfide bonds of TCR

[0689] We used the TCR crystal structure to guide our WuXiBody design. Unlike natural TCRs anchored on the surface membrane of T cells, soluble TCR molecules are less stable, although their 3D structure is very similar to antibody Fab. In fact, the instability of TCR in the dissolved state was a major obstacle to its crystal structure being difficult to elucidate (see Wang, Protein Cell, 5(9), pp.649–652(2014)). We adopted a scheme of introducing a pair of Cys mutations in the TCR constant region and found that it can significantly improve chain assembly and enhance expression.

[0690] The junction region connecting the antibody variable domain and the TCR constant domain, its relative fusion orientation and the junction region connecting Fc are all carefully fine-tuned. Because the TCR structure is very similar to the antibody Fab, we superimpose the antibody Fv homology model on the TCR variable region (PDB 4L4T). The superimposed structure shows that the antibody Fv is compatible with the TCR constant domain in structure. All relevant engineering parameters are designed based on this structural comparison and corresponding sequence.

[0691] 2. Preparation of the bispecific antibodies of the present disclosure

[0692] W3278 BsAbs as human IgG4 were produced in a handle-into-hole format (S. Atwell, JB Ridgway, JA Wells, P. Carter, Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol. 270, 26–35 (1997); C. Spiess, M. Merchant, A. Huang, et al. DG Yansura, JM Cheer, Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies. Nat. Biotechnol. 31, 753–758 (2013)). The human IgG4 Fc region sequence was designed with an S228P mutation. Anti-CD3 monoclonal antibodies were produced by immunizing mice with human CD3ε and CD3δ ECD proteins by an in-house protocol via hybridoma technology. The variable region sequence of the anti-CD20 arm is based on the sequence of Ofatumumab (clone 2F2 of PCT publication number WO2010083365A1) or rituximab (clone C2B8 of U.S. patent application US20140004037 A1). The sequences of the W3278 BsAb candidates are listed in Table 2, and their DNA sequences are synthesized at Genewiz (Shanghai) and cloned into a modified pcDNA3.3 expression vector. The expression vectors of the anti-CD20 arm and the anti-CD3 arm were co-transfected into Expi293 (Invitrogen-A14527) using the ExpiFectamine293 transfection kit (Invitrogen-A14524). The cells were cultured in an incubator at 37°C in a humidified atmosphere containing 8% CO2 on an orbital shaking platform rotating at 135 rpm in Expi293 expression medium (Invitrogen-A1435101). The culture supernatant was collected and protein was purified using a protein A column (GE Healthcare, 17543802). The protein concentration was measured by UV-Vis spectrophotometer (NanoDrop 2000, Thermo Scientific). Protein purity was estimated by SDS-PAGE and analytical HPLC-SEC. Figure 1 A schematic diagram of the W3278-BsAb candidate is depicted.

[0693] Table 2. Sequences of the heavy and / or light chains in each BsAb

[0694]

[0695]

[0696]

[0697]

[0698] Note: TCR sequences are in italics.

[0699] Further, in the bispecific antibody W3278-U2T3.F18R-1.uIgG4.SP, the inventors modified one or more amino acids in the natural glycosylation sites at positions 193, 182, 203, 206, and 207 of the light chain shown in SEQ ID NO: 44, preferably modifying the amino acid at position 193. In certain embodiments, the modification includes one or more of S193X, S182X, S203X, S206X, and S207X, wherein X is any amino acid other than serine (Ser) and / or threonine (Thr). In certain preferred embodiments, the modification is S193X, wherein X is alanine (Ala), glycine (Gly), proline (Pro), or valine (Val). The above mutation eliminates the O-glycosylation site, and the O-glycosylation type is O-sugar of Core1 configuration, with the structural formula of NeuAc-Gal-GalNAc or NeuAc-Gal-(NeuAc)GalNAc.

[0700] As described above, in the bispecific antibodies of the present invention, the natural glycosylation sites in the corresponding polypeptide chains are modified. Compared with the unmodified bispecific antibodies, the obtained bispecific antibodies are closer to natural antibodies, have significantly reduced immunogenicity, increased half-life, and improved drugability.

[0701] Example 3

[0702] In vitro characterization

[0703] 1. Cell Lines and Primary Cell Isolation

[0704] The following cell lines were cultured in complete medium (RPMI1640 supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin): Jurkat (CD3 + / CD20 - cells); Raji, Ramos and NAMALWA (CD20 + / CD3 -cells), SU-DHL-1(CD20 - / CD3 - cell).

[0705] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from heparinized venous blood from healthy normal donors by Ficoll-Paque PLUS (GE Healthcare-17-1440-03) density centrifugation. Primary human CD8 + T cells, and CD4 + T cells.

[0706] 2. Binding of W3278 BsAb to target cells

[0707] The binding of W3278 BsAb to target cells was determined by flow cytometry. Briefly, 1×10 5 Target cells / well (CD3 + / CD20 - Cells or CD20 + / CD3 - Cells) were incubated at 4°C for 60 minutes with serial dilutions of W3278 BsAb or human IgG4 isotype control antibody. After incubation, the cells were washed twice with cold 1% BSA / 1×PBS, and then AlexaFluor647-conjugated goat anti-human IgG Fc (Jackson-109-605-098) was added and incubated at 4°C for 30 minutes. After washing twice, the geometric mean fluorescence (MFI) of the stained cells was measured using a FACS Canto II blood cell counter (BD Biosciences). Wells containing no antibody or only fluorescent secondary antibodies were used to establish background fluorescence. The EC of cell binding was determined using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). 50 The values ​​were calculated using four-parameter nonlinear regression analysis. The FACS binding of W3278BsAb to target cells was shown in Figure 2 The binding EC50s are shown in Table 3 below.

[0708] Table 3. FACS binding EC50 of W3278 BsAbs and parental antibodies to cell surface targets

[0709]

[0710] To detect the simultaneous binding of W3278 BsAb to CD3 and CD20 expressing cells, 1×10 6 / ml Raji cells and 1×10 6 Jurkat cells were labeled with 50 nM Calcein-AM (Invitrogen-C3099) and 20 nM FarRed (Invitrogen-C34572) respectively. After washing with cold 1% BSA / 1XPBS, the labeled Raji and Jurkat cells were resuspended and mixed in a 1:1 ratio to a final concentration of 1×10 6 / ml. Use 1x10 5 100 cells / well mixed with 100 cells / well were plated, and then serial dilutions of W3278 BsAb were added. After incubation at 4°C for 60 minutes, the percentage of Calcein-AM and FarRed double positive cells was analyzed by FACS.

[0711] Figure 3 The results showed that W3278 lead BsAb exhibited dose-dependent simultaneous dual-target binding, which was more effective than BMK4.

[0712] 3. In vitro Cytotoxicity Assay

[0713] Determination of BsAb cytotoxicity via CD8 + T lymphocytes regulate the efficacy of tumor cell lysis. Briefly, freshly isolated human CD8 + T cells were cultured in complete medium containing 50 IU / ml recombinant human IL-2 and 10 ng / ml OKT-3 for 3 to 5 days. On the next day, target cells, Raji, Ramos, NAMALWA and SU-DHL-1 (1×10 6 Target cells (2x10 cells / ml) were labeled with 20 nM Far-Red (Invitrogen-C34572) in DPBS for 30 min at 37°C and then washed twice with assay buffer (RPMI 1640 medium without phenol red + 10% FBS). 4 / well) coated in 110μl / well containing effector CD8 + T cells (effector / target cell ratio of 5:1) and serial dilutions of BsAbs or hIgG4 isotype control were added to complete medium and incubated overnight at 37°C. Finally, propidium iodide (PI) (Invitrogen-P3566) was added and incubated at room temperature for 15 minutes before analysis by flow cytometry. The percentage of cytotoxicity was calculated using the following equation: Cytotoxicity % = 100*Far Red + PI+ / (Far Red + PI + +Far Red + PI - )*100%. The EC of in vitro cytotoxicity was determined using Prism four-parameter nonlinear regression analysis. 50 value.

[0714] Figure 4 The results showed that W3278 lead Ab "W3278-U2T3.F18R-1.uIgG4" did not kill CD20 negative SU-DHL-1 cells. Compared with BMK4, W3278 lead Ab "W3278-U2T3.F18R-1.uIgG4" more effectively induced cell killing of CD20 positive cells, and the killing efficiency EC50 increased in proportion to the cell surface CD20 expression level. BsAb-mediated cytotoxicity EC50 and maximum cytotoxicity (Max Cyto) % are shown in Table 4.

[0715] Table 4. Cytotoxicity EC50 and maximum cytotoxicity % of W3278 lead BsAb and BMK4 BsAb against different B cell lines

[0716]

[0717] 4. Cell Activation and Cytokine Release Assay

[0718] BsAb-mediated T cell activation was assessed by measuring the expression of CD69 or CD25 on effector cells by flow cytometry. Freshly isolated and purified CD4 + T cells and CD8 + T cells were tested as effector cells. 4 CD4 + or CD8 + T cells were plated in 110 μl / well complete medium containing serial dilutions of BsAbs or hIgG4 isotype control antibody at 1x10 4Raji or SU-DHL-1 cells / well in the presence of 37 ℃ 24 hours. After incubation, cells were washed twice with 1% BSA / 1XDPBS, and then stained with anti-human Ab group (FITC-labeled anti-human CD4 (BD Pharmingen-550628); PerCP-Cy5.5-labeled anti-human CD8 (BD Pharmingen-565310); PE-labeled anti-human CD69 (BD Pharmingen-555531) and APC-labeled anti-human CD25 (BD Pharmingen-555434)) at 4 ℃ for 30 minutes. T cell activation evaluated by CD69 or CD25 expression was analyzed by FACS. The EC50 of T cell activation was determined by using four-parameter nonlinear regression analysis.

[0719] In the absence of target cells, W3278 lead Ab did not induce T cell activation. Only in the presence of target cells, W3278 lead Ab induced CD4 + and CD8 + T cell activation, expressed by CD25 ( Figure 5A ) and CD69 expression ( Figure 5B ) and was more potent than BMK4. The activation EC50s are shown in Tables 5A and 5B.

[0720] Table 5A. EC50 of T cell CD25 expression mediated by W3278 BsAb and BMK4 BsAb in the presence of Raji cells

[0721]

[0722] Table 5B. EC50 of T cell CD69 expression mediated by W3278 BsAb and BMK4 BsAb in the presence of Raji cells

[0723]

[0724] For cytokine release (TNF-α and IL-2) assays, 5x10 4 Freshly isolated CD4 + T cells were plated in 110 μl / well complete medium containing serial dilutions of BsAbs or hIgG4 isotype control antibody at 1x10 4 The plates were incubated at 37° C. for 24 hours in the presence of 100 Raji or SU-DHL-1 cells / well. After 24 hours of incubation, the plates were centrifuged and the supernatants were collected and stored at −80° C. for cytokine concentration measurement by ELISA.

[0725] For TNF-α detection by ELISA, 96-well ELISA plates (Nunc MaxiSorp, ThermoFisher) were coated with 50 μl of capture antibody purified anti-human TNF (BD Pharmingen-51-26371E) in carbonate-bicarbonate buffer (20 mM Na2CO3, 180 mM NaHCO3, pH 9.2) at 4°C overnight. The next day, the plates were washed with wash buffer (1XPBST buffer, 0.05% Tween-20) and then blocked with 200 μl of assay diluent (PBS + 10% FBS). After blocking, 50 μl of test sample and recombinant human TNF standard (BD Pharmingen-51-26376E) were added, and the plates were incubated at room temperature for 2 hours. Binding of TNF-α to the plates was detected by detection antibody biotinylated anti-human TNF (BD Pharmingen-51-26372E). Streptavidin-HRP reagent (BD Pharmingen-51-9002813)) and tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) were used for the color reaction. Washing buffer was used between each step. The color reaction was stopped with 2M HCl after about 30 minutes. The plate was read using a multifunctional plate reader ( M5e) Measure the absorbance of each well at 450 nm.

[0726] Similarly, the IL-2 concentration in the culture supernatant was detected by ELISA. Anti-human IL-2 antibody mAb (R&D-MAB602) was used as the capture antibody, and biotinylated anti-human IL-2 antibody (R&D-BAF202) was used as the detection antibody.

[0727] like Figure 6 As shown in Table 6, in the presence of CD20 negative SU-DHL-1 cells, W3278 lead Ab did not induce cytokine release from T cells. Only in the presence of target Raji cells, W3278 lead Ab was able to induce lower levels of cytokine release compared to BMK4. And the EC50 window caused by W3278 Ab (shown as the EC50 ratio between cytokine release and cell killing) was greater than that of BMK4 (Table 6).

[0728] Table 6. BsAb-mediated CD4 + EC50 and maximum level of cytokine release from T cells and EC50 ratio of cytokine release to Raji cell killing

[0729]

[0730] 5. Serum stability test

[0731] Mix the antibody with freshly collected human serum and ensure that the serum ratio in the mixed sample is >95%. Aliquot the mixed sample and incubate at 37°C for 0-14 days. Figure 7 At each time point indicated, samples were snap frozen in liquid nitrogen and stored at -80°C prior to analysis. Binding of each sample to Raji or Jurkat cells was analyzed by FACS.

[0732] like Figure 7 As shown, human serum treated W3278 Ab and Jurkat ( Figure 7 A) and Raji( Figure 7 B) Binding of both cells was similar to that of freshly thawed Ab (day 0). These results indicate that W3278 Ab is stable in human serum for at least 14 days ( Figure 7 ).

[0733] 6.DSF determination and thermal stability test

[0734] DSF was determined using real-time fluorescence quantitative PCR (QuantStudio 7Flex, Thermo Fisher Scientific). Briefly, 19 μL of antibody solution was mixed with 1 μL of 62.5X SYPRO Orange solution (Invitrogen) and added to a 96-well plate (Biosystems). The plate was heated from 26°C to 95°C at a rate of 2°C / min, and the resulting fluorescence data was collected. The negative derivative of the fluorescence change relative to different temperatures was calculated, and the maximum value was defined as the melting temperature T h If the protein has multiple unfolding transition states, the first two T h , called T m1 and T m2 . T m1 Always interpreted as the formal melting temperature T m , to facilitate comparison between different proteins. Data acquisition and T h The calculation was performed automatically by the operating software (QuantStudio Real-Time PCR Software v1.3). T values ​​of W3278 Ab in different buffers m1 and T m2 The values ​​are shown in Table 7. The T m It is about 61°C, indicating good thermal stability of W3278 Ab.

[0735] Table 7. Thermal stability detected by DSF assay (Tm value is shown by Tm1 in the table)

[0736]

[0737] Example 4

[0738] In vivo anti-tumor efficacy

[0739] The in vivo anti-tumor efficacy of the antibody was tested in the Raji tumor model of PBMC humanized NOG mice. Female NOG mice aged 6-8 weeks (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used in the study. Raji tumor cells ( CCL-86 TM ) were cultured in monolayers in an incubator at 37°C with 5% CO2 and in 1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin. Tumor cells were routinely passaged twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation. Human PBMCs were isolated from heparinized whole blood from healthy donors using Ficoll-Paque Plus according to the manufacturer's instructions.

[0740] For the treatment model, each mouse was subcutaneously injected with premixed Raji tumor cells (2.0×10 6 ) and PBMC (3.0×10 6 When the average tumor volume reaches about 60 mm 3 At , the animals were randomized and received the first antibody injection. For efficacy studies, mice received the indicated amount of antibody intravenously twice a week for a total of 3 weeks. All procedures involving animal handling, care, and treatment in the study were performed in accordance with the guidelines approved by the WuXi AppTec Institutional Animal Care and Application Committee (IACUC) following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). For all tumor studies, mice were weighed and tumor growth was measured twice a week using calipers. Tumor volume was measured as 1 / 2 (length × width 2 ) estimate.

[0741] like Figure 8 As shown, W3278 lead Ab treatment exhibited dose-dependent antitumor activity, which was more effective than BMK4 ( Figure 8 A). The mice had normal body weight during the experiment ( Figure 8 B).

[0742] Example 5

[0743] Single-dose study with WBP3278 bispecific antibody in naive cynomolgus monkeys

[0744] To determine whether treatment with the WBP3278 bispecific antibody can deplete circulating B cells in primates and determine whether any unexpected toxicity is caused, the inventors conducted an exploratory non-GLP pharmacology study in cynomolgus monkeys (Macaca Fascicularis). Four male cynomolgus monkeys (age 3-4 years old, weighing about 4 kg) for immunization for the first time were provided by Guangdong Zhaoqing Chuangyao Biotechnology Co., Ltd. All steps related to the handling, care and treatment of animals in the study were carried out in accordance with the guidelines approved by the PharmaLegacy Laboratory Institutional Animal Care and Use Committee (IACUC) following the guidance of the Assessment and Accreditation Committee for Laboratory Animal Care (AAALAC).

[0745] Four animals were divided into two groups (2 animals in each group) and WBP3278 lead antibody (i.e., W3278-U2T3.F18R-1.uIgG4, hereinafter referred to as WBP3278 lead Ab) was administered once by slow intravenous injection within 60 seconds at 1 mg / kg (Group 1) and 10 mg / kg (Group 2). The levels of peripheral blood circulating B cells and T cells were monitored by FACS for 4 weeks for the following lymphocytes: B lymphocytes (CD45 + / CD20 + ); T lymphocytes (CD45 + / CD3 + ); CD4 + T lymphocytes (CD4 + / CD45 + / CD3 + ) and CD8 + T lymphocytes (CD8 + / CD45 + / CD3 + ). By using BD TM The levels of circulating inflammatory cytokines were analyzed by Cytometric Bead Array (CBA) non-human primate Th1 / Th2 cytokine kit (BD Bioscience, Cat: 557800). Treatment with WBP3278 lead Ab resulted in immediate and complete depletion of circulating B cells that lasted for at least 4 weeks ( Fig. 9 After WBP3278 lead Ab treatment, the levels of circulating T cells were also initially reduced, returning to baseline levels or slightly elevated levels after 72 hours and persisting for at least 4 weeks ( Fig. 10A After 72 hours, CD8+T cell levels increased ( Fig. 10B ), CD4 + T cell levels returned to normal levels ( Fig. 10C), and all lasted for at least 4 weeks. After treatment with WBP3278lead Ab, a rapid increase in circulating cytokine levels was observed, and all cytokines returned to normal levels after 24 hours ( Fig.11 ).

[0746] The concentration of WBP3278 lead Ab in serum was determined by ELISA. Briefly, the ELISA plate was coated with anti-human IgG (SouthernBiotech, #2049-01) and then serial dilutions of serum samples were added. The binding signal was detected with goat anti-human IgG biotin (SouthernBiotech, #2049-08) and streptavidin-HRP (Life, #SNN1004). The plate was read using a multiwell plate reader ( Absorbance was measured at (450-540) nm using a 450 nm (M5e). Non-compartmental pharmacokinetic analysis of WBP3278 leadAb concentrations in cynomolgus monkeys was performed using PhoenixWinNonlin software (version 8.1, Pharsight, Mountain View, CA). PK parameters were obtained using the linear / logarithmic trapezoidal rule. Individual BLQs were excluded when calculating mean concentrations. Normal dose levels and nominal sampling times were used when calculating all pharmacokinetic parameters. A summary of the PK parameters is listed in Table 8 and is shown in Table 8. Fig.12 It can be seen that when the dose increases from 1 mg / kg to 10 mg / kg, the systemic exposure C0 increases from 19.9 μg / mL to 282 μg / mL (about 14 times), and AUC 0-last The serum half-life (T) of WBP3278 bispecific antibody increased from 259 μg.h / mL to 5788 μg.h / mL (about 22 times). 1 / 2 ) were 43.3 hours and 89.8 hours at 1 mg / kg and 10 mg / kg, respectively.

[0747] Table 8. Summary of PK parameters of WBP3278 lead Ab

[0748] PK parameters 1mg / kg (average) 10mg / kg (average) <![CDATA[C0(μg / mL)]]> 19.9 282 <![CDATA[T 1 / 2 (h)]]> 43.3 89.8 <![CDATA[AUC 0-last (μg.h / mL)]]> 259 5788

[0749] In addition, cage-side observations during the experiment showed that no unexpected toxicity was observed for both high and low dose levels of WBP3278 lead Ab (data not shown).

[0750] The results of this experiment show that WBP3278 lead Ab can effectively eliminate B cells in the body, without adverse reactions such as cytokine storm, and has a sufficient half-life in crab-eating monkeys. These experimental results support the advancement of WBP3278 lead Ab into preclinical drug development.

[0751] Examples of Formulations

[0752] Size exclusion high performance liquid chromatography (SEC-HPLC): used to determine the purity of the antibody in the sample. Example 6 used an Agilent 1260 high performance liquid chromatograph, a TSK G3000SWXL (TOSOH) gel chromatography column, eluted with a 50 mM phosphate buffer + 300 mM sodium chloride (pH 6.8) buffer as the mobile phase, and the detection wavelength was 280 nm. Examples 7, 8.1, 8.2 and 10 used a Waters I-Class high performance liquid chromatograph, an Agilent AdvanceBio SEC The gel chromatography column was eluted with a buffer solution of 17.8 mM sodium dihydrogen phosphate + 32.2 mM sodium dihydrogen phosphate + 200 mM L-arginine hydrochloride as the mobile phase, and the detection wavelength was 280 nm. The percentage of polymer, main peak and degradation fragment was calculated by area normalization method.

[0753] Differential scanning calorimetry (DSC): Examples 6-7 used SYS12907 (Malvern) to analyze the inflection point temperature (Tmonset) and unfolding temperature (Tm) of the sample, and the sample was diluted to a concentration of 1 mg / mL, and the program was as follows: the scanning start temperature was 10°C, the scanning end temperature was 95°C, and the heating rate was 200°C / h. Example 10 used the protein stability analyzer PrometheusNT.48 (NanoTemper) to analyze the inflection point temperature (Tmonset) and melting temperature (Tm) of the sample, and 10 μL of the sample was taken and placed in the sample chamber, and the program was as follows: the scanning start temperature was 25°C, the scanning end temperature was 95°C, and the heating rate was 0.3°C / min.

[0754] Example 6 DynaproplateReaderII (Wyatt) was used to detect the aggregation temperature (Tagg) of the sample. 20 μL of the sample was added to a 384-well sample plate, and 20 μL of paraffin oil was added to the sample. The 384-well sample plate was centrifuged to remove bubbles. The procedure was as follows: the temperature was raised from 25°C to 60°C, the scanning time was 5 seconds, and the scanning was performed 5 times. Example 10 DynaproplateReaderⅢ (Wyatt) was used to detect the aggregation temperature (Tagg) of the sample. 30 μL of the sample was added to a 384-well sample plate, the plate was sealed with a sealing film, and the 384-well sample plate was centrifuged to remove bubbles. The procedure was as follows: the temperature was raised from 25°C to 85°C, the scanning time was 5 seconds, and the scanning was performed 5 times.

[0755] Modulated differential scanning calorimetry (mDSC): The glass transition temperature (Tg') of the samples during freeze-drying was analyzed using DSC Q2000 (TA Instruments) with the following procedure: cooling to -60 °C, holding for 5 min, and heating to 20 °C at a heating rate of 5 °C / min.

[0756] The number of insoluble particles was analyzed using a particle detector GWJ-8 (Tianjin Tianda Tianfa GWJ-8 Technology Co., Ltd.); the number of MFI particles was analyzed using a microfluidic imaging particle analysis system MFI5100 (ProteinSimple), and the data were analyzed using the instrument's supporting MVAS software.

[0757] The moisture content of the samples was detected using a moisture titrator C30 (METTLER TOLEDO).

[0758] Appearance: Wipe the sample bottle clean and place it under a clarity tester. Adjust the illumination to 1500 lx and visually inspect the appearance of the solution and visible foreign matter against a black and white background respectively.

[0759] Example 6

[0760] Buffer system and pH value

[0761] By dialysis, the WBP3278 lead Ab was replaced into the target buffer at a protein concentration of 2 mg / mL. The solution was filtered using a 0.22 μm PVDF filter in a biosafety cabinet and dispensed into 2 mL vials at a specification of 2 mg / mL / vial, stoppered and capped. The stability test was conducted at 35°C, and the Tm value, Tagg value and SEC-HPLC test results are shown in Table 9, Table 10 and Table 11, respectively.

[0762] Table 9. Tm value detection results

[0763]

[0764] Note: “-” means no data was detected

[0765] Table 10. Tagg value detection results

[0766]

[0767] Table 11. SEC-HPLC test results

[0768]

[0769]

[0770] Note: "T0" means time 0; "ND" means no data detected; (+X) means an increase of X relative to T0, (-X) means a decrease of X relative to T0, for example, polymer = 17.9 (+17.1), means the polymer is 17.9%, an increase of 17.1% relative to T0

[0771] Example 7

[0772] Sugar and surfactants

[0773] Prepare 1 kg of 10 mM citric acid-sodium citrate buffer (pH 6.2), weigh 0.282 g of citric acid monohydrate (C6H8O7·H2O), 2.547 g of sodium citrate dihydrate (C6H5Na3O7·2H2O), add water to make up to 1 kg, and filter the buffer.

[0774] By dialysis and liquid exchange, WBP3278 lead Ab was replaced into the 10mM citric acid-sodium citrate buffer prepared above, and sucrose mother solution, dihydrate trehalose mother solution or mannitol mother solution were added respectively, and Tween 80 mother solution was added to prepare the target formulation, the protein concentration was 2mg / mL, and after filtering with a 0.22μm PVDF filter in a biological safety cabinet, it was dispensed into 4mL cillin bottles with a specification of 2mg / mL / bottle, stoppered and capped. Among them, 8% (w / v) sucrose is 80mg / ml sucrose; 8.8% (w / v) dihydrate trehalose is 88mg / ml dihydrate trehalose; 0.02% (w / v) Tween 80 is 0.2mg / mL Tween 80. The Tm value test results, SEC-HPLC and pH test results of the three formulations are shown in Table 12 and Table 13, respectively.

[0775] Table 12. Tm value detection results

[0776]

[0777] Table 13. SEC-HPLC and pH test results

[0778]

[0779]

[0780] Note: “T0” means time 0; the freezing and thawing conditions are from -70℃ to room temperature

[0781] Example 8.1

[0782] Sugar and surfactants

[0783] By dialysis, the WBP3278 lead Ab was replaced into 10 mM citric acid-sodium citrate buffer (prepared by the preparation method shown in Example 7), and sucrose mother solution, dihydrate trehalose mother solution or mannitol mother solution were added respectively, and Tween 80 mother solution was added to prepare the target formulation, the protein concentration was 2 mg / mL, and after filtering with a 0.22 μm PVDF filter membrane in a biological safety cabinet, it was divided into 4 mL vials, the specification was 2 mg / mL / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. The freeze drying procedure was pre-freezing, primary drying and secondary drying, and the steps were shown in Table 14. The three formulations were reconstituted with water, and the appearance, reconstitution time and water content after reconstitution were shown in Table 15; the Tg' value and SEC-HPLC test results are shown in Table 16 and Table 17, respectively.

[0784] Table 14. Lyophilization steps

[0785]

[0786] Table 15. Results of the appearance, reconstitution time and moisture content of the lyophilized formulations after reconstitution

[0787]

[0788] Note: “T0” means 0 time

[0789] Table 16. Tg' value test results

[0790]

[0791]

[0792] Table 17. SEC-HPLC and pH test results

[0793]

[0794] Note: “T0” means 0 time

[0795] Example 8.2

[0796] Sugar and surfactants

[0797] By ultrafiltration, WBP3278 lead Ab was replaced into 10 mM citric acid-sodium citrate buffer (see the preparation method shown in Example 7), sucrose mother solution or trehalose dihydrate mother solution was added, and Tween 80 mother solution was added to prepare the target formulation, the protein concentration was 2 mg / mL, and after secondary filtration with 0.22 μm PVDF filter membrane, it was divided into 6 mL vials with a specification of 2 mg / mL / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. The steps are shown in Table 14. The SEC-HPLC test results of the two formulations are shown in Table 18. In addition, the MFI particles and insoluble particles of the two formulations were within the normal range, but the MFI particles in the range of 2≤X<5μm in the formulation containing sucrose were much more than that in the formulation containing trehalose, and the insoluble particles in the range of 2≤X<5μm were also slightly more than that in the formulation containing trehalose; after being placed at 40℃ for 4 weeks, 25℃ for 6 months, and 2-8℃ for 6 months, the particles of the two formulations did not change significantly.

[0798] Table 18. SEC-HPLC test results

[0799]

[0800] Note: “T0” means 0 time

[0801] Example 9

[0802] Biological activity

[0803] The biological activity was determined by in vitro T cell activation assay. The target cells were Raji (CD3 - 、CD20 + Human B cell line), the effector cells were engineered Jurkat expressing luciferase (CD3 + 、CD20 - Human T cell line), when the CD3 and CD20 binding parts of WBP3278 lead Ab bind to Raji cells and Jurkat cells respectively, Jurkat cells are activated and downstream signal transduction is induced, luciferase is expressed, and the fluorescence signal is detected by the detection reagent Bio-Glo. The reference product (a sample of a drug composition with a representative batch of the process) and the test product (a sample of the drug composition to be tested) are diluted in a gradient (initial concentration of 9000ng / mL, 4-fold gradient dilution, a total of 11 dilution gradients), and then the dilution is added to a 96-well cell culture plate at 25μL / well. Raji and Jurkat cells grown to the logarithmic phase are adjusted to 2×10 6cells / mL, mix at equal volumes, add 50 μL / well to the above 96-well cell culture plate, and incubate in a 37°C, 5% CO2 carbon dioxide cell culture incubator for 4-5 hours. Add 75 μL / well Bio-Lite Luciferase reagent and incubate at room temperature in the dark for 3-5 minutes. Read the RLU value using the chemiluminescence detection module of the multi-function plate reader.

[0804] Biological activity of the test sample (%) = (EC50 value of the reference sample / EC50 value of the test sample) × 100%

[0805] The experimental results showed that after being placed at 40°C for 4 weeks, 25°C for 6 months, and 2-8°C for 6 months, the WBP3278 lead Ab in the lyophilized formulation still maintained good biological activity.

[0806] Table 19. Biological activity test results

[0807]

[0808] Note: “T0” means 0 time

[0809] Example 10

[0810] By ultrafiltration, WBP3278 lead Ab was replaced into citric acid-sodium citrate buffer (prepared by the preparation method shown in Example 7), osmotic pressure regulator and / or stabilizer mother solution was added, and Tween 80 mother solution was added to prepare the target formulation (as shown in Table 20), and after secondary filtration with 0.22 μm PVDF filter membrane, it was dispensed into 6 mL medium borosilicate glass tube injection bottles, with a specification of 1 mL / bottle, stoppered and capped. The test results of Tm and Tagg values ​​of different formulations are shown in Table 21, and the test results of appearance and SEC-HPLC are shown in Table 22. In addition, the biological activities of all formulations are within the normal range (see Example 9 for the detection method).

[0811] Table 20. Ingredients of formulations

[0812]

[0813] Note: / indicates none

[0814] Table 21. Tm and Tagg value detection results

[0815]

[0816] Table 22. Appearance and SEC-HPLC test results

[0817]

[0818] Note: “\” indicates not tested yet

[0819] Those skilled in the art will further appreciate that the present disclosure can be implemented in other specific forms without departing from its spirit and core attributes. In the foregoing description, the present disclosure discloses only exemplary embodiments, and it should be understood that other variations are also included within the scope of the present disclosure. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. On the contrary, reference should be made to the appended claims regarding the scope and content of the present disclosure.

Claims

1. A liquid formulation comprising a bispecific anti-CD3 / CD20 polypeptide complex and a buffer, wherein: The buffer comprises one or more combinations of histidine buffer, succinate buffer and citrate buffer.

2. The liquid formulation according to claim 1, wherein the buffer comprises one or more of histidine-histidine hydrochloride buffer, succinic acid-sodium succinate buffer, and citric acid-sodium citrate buffer, preferably, the buffer is citric acid-sodium citrate buffer. 3 . The liquid formulation according to claim 1 , wherein the concentration of the buffer is about 1 mM to about 30 mM, preferably about 5 mM to about 20 mM, more preferably about 10 mM.

4. The liquid formulation according to any one of claims 1 to 3, wherein the concentration of the bispecific anti-CD3 / CD20 polypeptide complex is about 0.1 mg / ml to about 50 mg / ml, preferably about 0.5 mg / ml to 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, more preferably about 2 mg / ml to about 10 mg / ml, or about 2 mg / ml to about 5 mg / ml.

5. The liquid formulation according to any one of claims 1 to 4, wherein the pH of the liquid formulation is about 5.0 to about 7.5, or about 5.0 to about 6.5, preferably about 6.0 to about 6.5, more preferably about 6, about 6.1 or about 6.

2.

6. The liquid formulation according to any one of claims 1 to 5, wherein the liquid formulation further comprises an osmotic pressure regulator and / or a stabilizer; Preferably, the osmotic pressure regulator and / or stabilizer comprises sugar; More preferably, the sugar is trehalose or sucrose; Most preferably, the sugar is trehalose.

7. The liquid formulation according to claim 6, wherein the concentration of the sugar is 150 mM to about 320 mM, preferably about 180 mM to about 265 mM, about 230 mM to about 240 mM, or about 230 mM to about 235 mM, more preferably about 233 mM or about 232.6 mM.

8. The liquid formulation according to claim 6, wherein the osmotic pressure regulator and / or stabilizer further comprises one or more of sodium chloride, arginine-hydrochloride, glycine or proline; Preferably, the concentration of sodium chloride, arginine-HCl, glycine or proline is about 10 mM to about 300 mM, preferably about 20 mM to about 200 mM, or about 50 mM to about 150 mM, more preferably about 100 mM to about 110 mM, or about 100 mM to about 105 mM.

9. The liquid formulation according to any one of claims 1 to 8, wherein the liquid formulation further comprises a surfactant; Preferably, the surfactant is polysorbate 80 or polysorbate 20, preferably polysorbate 80; Preferably, the concentration of the surfactant is about 0.001% to about 0.1% (w / v), preferably about 0.005% to about 0.08% (w / v), or about 0.01% to about 0.04% (w / v), more preferably about 0.02% (w / v).

10. The liquid formulation according to any one of claims 1 to 9, wherein the liquid formulation comprises: (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex, (b) about 1 mM to about 30 mM citrate buffer, (c) about 150 mM to about 320 mM of a sugar, and optionally, one or more of about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and (d) from about 0.001% to about 0.1% (w / v) of a surfactant, wherein the pH of the liquid formulation is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5; Optionally, the liquid formulation comprises: (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex, (b) about 1 mM to about 30 mM citrate buffer, (c) about 150 mM to about 320 mM trehalose, and optionally, one or more of about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and (d) about 0.001% to about 0.1% (w / v) polysorbate 80, wherein the pH of the liquid formulation is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5; Optionally, the liquid formulation comprises: (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex, (b) about 5 mM to about 20 mM citrate buffer, (c) about 180 mM to about 265 mM trehalose, and optionally, one or more of about 20 mM to about 200 mM sodium chloride, about 20 mM to about 200 mM arginine-hydrochloride, about 20 mM to about 200 mM glycine, or about 20 mM to about 200 mM proline, and (d) about 0.005% to about 0.08% (w / v) polysorbate 80, wherein the pH of the liquid formulation is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5; Optionally, the liquid formulation comprises: (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex, (b) about 5 mM to about 20 mM citric acid-sodium citrate buffer, (c) about 180 mM to about 265 mM trehalose, and optionally, one or more of about 20 mM to about 200 mM sodium chloride, about 20 mM to about 200 mM arginine-hydrochloride, about 20 mM to about 200 mM glycine, or about 20 mM to about 200 mM proline, and (d) about 0.005% to about 0.08% (w / v) polysorbate 80, The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.

5.

11. The liquid formulation according to any one of claims 1 to 9, wherein the liquid formulation comprises: (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex, (b) about 1 mM to about 30 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer, (c) about 150 mM to about 320 mM of a sugar, and one or more of about 10 mM to about 300 mM sodium chloride, about 10 mM to about 300 mM arginine-hydrochloride, about 10 mM to about 300 mM glycine, or about 10 mM to about 300 mM proline, and (d) from about 0.001% to about 0.1% (w / v) of a surfactant, wherein the pH of the liquid formulation is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5; Optionally, the liquid formulation comprises: (a) about 0.5 mg / ml to about 20 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex, (b) about 5 mM to about 20 mM histidine-histidine hydrochloride or citric acid-sodium citrate buffer, (c) about 180 mM to about 265 mM trehalose or sucrose, and about 20 mM to about 200 mM sodium chloride and / or about 20 mM to about 200 mM arginine-hydrochloride, and (d) about 0.005% to about 0.08% (w / v) polysorbate 20 or polysorbate 80, wherein the pH of the liquid formulation is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5; Optionally, the liquid formulation comprises: (a) about 0.1 mg / ml to about 50 mg / ml of a bispecific anti-CD3 / CD20 polypeptide complex, (b) about 1 mM to about 30 mM citrate buffer, (c) from about 150 mM to about 320 mM trehalose, and from about 10 mM to about 300 mM sodium chloride, and (d) about 0.001% to about 0.1% (w / v) polysorbate 80 or polysorbate 20, wherein the pH of the liquid formulation is from about 5.0 to about 7.5, preferably from about 6.0 to about 6.5; Optionally, the liquid formulation comprises: (a) about 0.5 mg / ml to about 20 mg / ml of the bispecific anti-CD3 / CD20 polypeptide complex, (b) about 5 mM to about 20 mM citrate buffer, (c) about 180 mM to about 265 mM trehalose, and about 20 mM to about 200 mM sodium chloride, and (d) about 0.005% to about 0.08% (w / v) polysorbate 80 or polysorbate 20, The pH of the liquid formulation is about 5.0 to about 7.5, preferably about 6.0 to about 6.

5.

12. A lyophilized preparation containing a bispecific anti-CD3 / CD20 polypeptide complex, the lyophilized preparation being obtained by freeze-drying the liquid formulation according to any one of claims 1 to 11; or The lyophilized agent can be reconstituted to form the liquid formulation according to any one of claims 1 to 11.

13. A pharmaceutical composition containing a bispecific anti-CD3 / CD20 polypeptide complex, wherein the pharmaceutical composition is a solution obtained by reconstituting the lyophilized agent according to claim 12.

14. The liquid formulation of any one of claims 1 to 11, the lyophilized agent of claim 12, or the pharmaceutical composition of claim 13, wherein the bispecific anti-CD3 / CD20 polypeptide complex comprises a first antigen binding moiety associated with a second antigen binding moiety, wherein: The first antigen binding moiety comprises: a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), in: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between C1 and C2, and the non-native interchain bond is capable of stabilizing the dimer, and The second antigen binding moiety comprises: a second heavy chain variable domain (VH2) of a second antibody, operably linked to the antibody heavy chain CH1 domain, and a second light chain variable domain (VL2) of a second antibody operably linked to an antibody light chain constant (CL) domain, wherein: one of said first and said second antigen binding moiety is an anti-CD3 binding moiety and the other is an anti-CD20 binding moiety, The anti-CD3 binding portion is derived from an anti-CD3 antibody and comprises: a) a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 13, b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 14, c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 15, d) a kappa light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 16, e) a kappa light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 17, and f) a kappa light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 18, The anti-CD20 binding portion is derived from an anti-CD20 antibody, which comprises: a) a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 19, b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 20, c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 21, d) a kappa light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 22, e) a kappa light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 23, and f) a kappa light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 24.

15. The liquid formulation, lyophilized agent, or pharmaceutical composition according to claim 14, wherein: The anti-CD3 binding portion of the bispecific anti-CD3 / CD20 polypeptide complex comprises a heavy chain variable domain sequence selected from SEQ ID NOs: 25 and 27 and a light chain variable domain sequence selected from SEQ ID NOs: 26 and 28, or comprises a heavy chain variable domain sequence containing a C-terminal deleted amino acid SS selected from SEQ ID NOs: 25 and 27 and a light chain variable domain sequence selected from SEQ ID NOs: 26 and 28; The anti-CD20 binding portion of the bispecific anti-CD3 / CD20 polypeptide complex comprises a heavy chain variable domain sequence selected from SEQ ID NOs: 29 and 31 and a light chain variable domain sequence selected from SEQ ID NOs: 30 and 32.

16. The liquid formulation, lyophilized agent, or pharmaceutical composition according to claim 14 or 15, wherein: The bispecific anti-CD3 / CD20 polypeptide complex comprises the first antigen binding portion connected to a first dimerization domain, the second antigen binding portion connected to a second dimerization domain, and the first and second dimerization domains are associated; Optionally, the association in the bispecific anti-CD3 / CD20 polypeptide complex is achieved via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge or a hydrophobic-hydrophilic interaction or a combination thereof; Optionally, the first and second dimerization domains in the bispecific anti-CD3 / CD20 polypeptide complex are different and are associated in a manner that is not conducive to homodimerization and / or is conducive to heterodimerization; Optionally, the first and the second dimerization domains in the bispecific anti-CD3 / CD20 polypeptide complex can associate into a heterodimer via handle-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction or increased flexibility.

17. The liquid formulation, lyophilized agent, or pharmaceutical composition according to claim 16, wherein the first and / or the second dimerization domain in the bispecific anti-CD3 / CD20 polypeptide complex comprises at least a portion of an antibody hinge region, which is optionally derived from IgG1, IgG2 or IgG4; Optionally, the first and / or the second dimerization domain in the bispecific anti-CD3 / CD20 polypeptide complex comprises an antibody CH2 domain and / or an antibody CH3 domain; Optionally, the first dimerization domain in the bispecific anti-CD3 / CD20 polypeptide complex is operably linked to the first TCR constant region (C1) at the third engagement domain; Optionally, the second dimerization domain in the bispecific anti-CD3 / CD20 polypeptide complex is operably linked to the heavy chain variable domain of the second antigen binding portion.

18. The liquid formulation, lyophilized agent, or pharmaceutical composition according to any one of claims 14 to 17, wherein the bispecific anti-CD3 / CD20 polypeptide complex comprises a combination of four polypeptide sequences: a) SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; b) SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; c) SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44; d) SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48; or e) SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52.

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