Stable anti-cd79b immunoconjugate formulations

Abstract

The present invention relates to stable anti-CD79B immunoconjugate formulations, and specifically provides a stable pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant. The present invention also provides methods of using such compositions to treat cancer.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980025490.1, filed on April 12, 2019, entitled "Stable Anti-CD79B Immunocytic Conjugate Preparation".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 657,185, filed April 13, 2018, the entire disclosure of which is incorporated herein by reference.

[0004] Submitting sequence lists as ASCII text files

[0005] The following ASCII text file is incorporated herein by reference in its entirety: Computer-readable format (CRF) of sequence lists (filename: 146392044340SEQLIST.TXT, date of record: April 9, 2019, size: 12 KB). Technical Field

[0006] This disclosure relates to stable pharmaceutical compositions comprising an anti-CD79b immunoconjugate and a surfactant. This disclosure also provides methods of treating cancer using such compositions. Background Technology

[0007] In commercial settings, intravenous (IV) administration via infusion bags is the most common route of delivery for biologics. To support the delivery and compatibility of biologics such as immunoconjugates via intravenous (IV) administration, it is necessary to design appropriate therapeutic formulations that remain stable after dilution in the infusion bag, during transport, and throughout the administration process (Bardin, C. et al., Annales pharmaceutiques francaises 69 (2011) 221-231).

[0008] One challenge in biologics administration, particularly using IV infusion bags, is that the structure and materials of the infusion solution can destabilize the environment of therapeutic proteins. Furthermore, proteins are subjected to interfacial stresses within the IV infusion bag. Protein adsorption at solid-liquid and air-water interfaces can cause surface protein denaturation, leading to protein aggregation (Shieh, I. et al., *Molecular Pharmaceutics* 12 (2015): 3184-93; Sreedhara, A. et al., *Pharmaceutical Science* 101 (2012): 21-30). The agitation of the infusion bag further complicates IV administration by causing continuous regeneration of the air-liquid interface, resulting in repeated protein degradation over time. Aggregation caused by unstable solution conditions or interfacial stresses in IV infusion bags can have serious adverse effects on the product quality, potency, and immunogenicity of biologics.

[0009] The adverse effects of intravenous administration on biologics can be partially mitigated by the use of surfactants in formulation development. Nonionic surfactants such as polysorbate-20 (PS20) or polysorbate-80 (PS80) are commonly used in protein formulations to protect and stabilize molecules from air-water interfacial stress (Kerwin, BA. J. Pharm. Sci. 97 (2008) 2924-2935). Surfactants can protect drug products (DPs) from surface-induced stress by competing with proteins at air-water and solid-water interfaces (Kerwin, BA. J. Pharm. Sci. 97 (2008) 2924-2935). Furthermore, surfactants reduce the surface tension of the system (Cleland, JL. et al., Critical reviews in therapeutic drugcarrier systems 10 (1993) 307-377). However, a key drawback of using nonionic surfactants at concentrations suitable for IV bag delivery is that prolonged exposure of biologics to surfactants can lead to oxidation of specific amino acid residues, thereby reducing efficacy. (Lam X et al., *Pharmaceutical Research* (2011) 28:2543-2555.)

[0010] Importantly, prior to intravenous administration, biologics must also have a long and stable shelf life under storage conditions during which the structure and activity of the therapeutic protein remain unaffected. For example, liquid formulations of antibody-drug conjugates (ADCs) employing linkers may experience acid-catalyzed hydrolysis of the linker during storage. Such instability can lead to premature release of the drug compound after intravenous administration, adversely affecting the pharmacokinetics and safety of the biologic.

[0011] Therefore, there is a need to develop a stable pharmaceutical composition that remains stable during intravenous administration and has a long shelf life under storage conditions. This disclosure meets this need and provides other related advantages. Summary of the Invention

[0012] In one aspect, this disclosure provides a pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant, wherein the concentration of the surfactant is at least 0.06% w / v (i.e., 0.6 mg / mL) and wherein the anti-CD79b immunoconjugate comprises the formula:

[0013]

[0014] in:

[0015] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (e.g., between about 2 and about 5). Between, for example, approximately 3.5).

[0016] In some embodiments, the concentration of the anti-CD79b immunoconjugate is about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 30 mg / mL, or about 10 mg / mL to about 20 mg / mL.

[0017] In some embodiments, the concentration of the anti-CD79b immunoconjugate is from about 10 mg / mL to about 20 mg / mL. In one embodiment, the concentration of the anti-CD79b immunoconjugate is about 10 mg / mL. In another embodiment, the concentration of the anti-CD79b immunoconjugate is about 20 mg / mL.

[0018] In some embodiments, the concentration of the anti-CD79b immunoconjugate is 5 mg / mL to 60 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL, or 10 mg / mL to 20 mg / mL.

[0019] In some embodiments, the concentration of the anti-CD79b immunoconjugate is from 10 mg / mL to 20 mg / mL. In one embodiment, the concentration of the anti-CD79b immunoconjugate is 10 mg / mL. In another embodiment, the concentration of the anti-CD79b immunoconjugate is 20 mg / mL.

[0020] In some embodiments, the concentration of the surfactant is between about 0.06% w / v (i.e., 0.6 mg / mL) and about 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the concentration of the surfactant is at least about 0.06% w / v (i.e., 0.6 mg / mL). In another embodiment, the concentration of the surfactant is at least about 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the concentration of the surfactant is at least 0.06% w / v. In another embodiment, the concentration of the surfactant is at least 0.12% w / v. In some embodiments, the concentration of the surfactant is 0.06% w / v. In another embodiment, the concentration of the surfactant is 0.12% w / v.

[0021] In some embodiments, the concentration of the anti-CD79b immunoconjugate is about 10 mg / mL, and the concentration of the surfactant is about 0.06% w / v (i.e., 0.6 mg / mL). In some embodiments, the concentration of the anti-CD79b immunoconjugate is about 20 mg / mL, and the concentration of the surfactant is at least about 0.12% w / v. In some embodiments, the concentration of the anti-CD79b immunoconjugate is about 20 mg / mL, and the concentration of the surfactant is about 0.12% w / v (i.e., 1.2 mg / mL).

[0022] In some embodiments, the concentration of the anti-CD79b immunoconjugate is 10 mg / mL, and the concentration of the surfactant is 0.06% w / v (i.e., 0.6 mg / mL). In some embodiments, the concentration of the anti-CD79b immunoconjugate is 20 mg / mL, and the concentration of the surfactant is at least 0.12% w / v. In some embodiments, the concentration of the anti-CD79b immunoconjugate is 20 mg / mL, and the concentration of the surfactant is 0.12% w / v.

[0023] In some embodiments according to (or applied to) any of the above embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C-8°C for at least about 24, 48, or 72 hours.

[0024] In some embodiments of this disclosure, the surfactant described herein is a nonionic surfactant. In one exemplary embodiment, the surfactant is selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188 (P188), N-octyl-β-D-glucopyranoside (OG), and combinations thereof. In one specific embodiment, the surfactant is PS20. In another specific embodiment, the surfactant is PS80.

[0025] In some embodiments of this disclosure, the composition further comprises a buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a succinate buffer. In one specific embodiment, the succinate buffer is a sodium succinate buffer. In some embodiments, the concentration of the sodium succinate buffer is from about 10 mM to about 200 mM. In some embodiments, the concentration of the sodium succinate buffer is from 10 mM to 200 mM. In one embodiment, the concentration of the sodium succinate buffer is about 10 mM. In another embodiment, the concentration of the sodium succinate buffer is 10 mM.

[0026] In some embodiments, the compositions of this disclosure have a pH of about 5.0 to about 6.0. In specific embodiments, the buffer solution has a pH of 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, or about 6.0. In one embodiment, the compositions of this disclosure have a pH of about 5.3. In some embodiments, the compositions of this disclosure have a pH of 5.0 to 6.0. In specific embodiments, the buffer solution has a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In one embodiment, the compositions of this disclosure have a pH of 5.3.

[0027] In some embodiments of this disclosure, the composition further comprises sugar. In some embodiments, the sugar concentration is from about 100 mM to about 260 mM. In some embodiments, the sugar concentration is from 100 mM to 260 mM. In some embodiments, the sugar is selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, dextran 40, and trehalose.

[0028] In one specific embodiment, the sugar is sucrose. In one embodiment of this disclosure, the concentration of sucrose is about 120 mM. In another embodiment, the concentration of sucrose is 120 mM.

[0029] In some embodiments, the compositions disclosed herein are lyophilized products (such as lyophilized blocks). In some embodiments, the lyophilized compositions are contained in a bottle (e.g., a 20 mL glass bottle).

[0030] In one aspect, this disclosure provides a pharmaceutical composition prepared by lyophilization of a liquid formulation comprising 20 mg / mL of an anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v of polysorbate 20, and 120 mM sucrose, wherein the liquid formulation has a pH of 5.3, and wherein the anti-CD79b immunoconjugate has the formula:

[0031]

[0032] in:

[0033] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (e.g., between about 2 and about 5). Between, for example, approximately 3.5).

[0034] In some embodiments of this disclosure, the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0035] A pharmaceutical composition is also provided, which is prepared by lyophilization of a liquid formulation comprising 20 mg / mL of an anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v polysorbate 20, and 120 mM sucrose, wherein the liquid formulation has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0036]

[0037] Wherein, Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5 (such as about 3.5). In some embodiments, the pharmaceutical composition is a lyophilized block.

[0038] In some embodiments, the pharmaceutical compositions of this disclosure remain stable for approximately 60 months when stored protected from light at 5°C ± 3°C. In another embodiment, the pharmaceutical compositions of this disclosure remain stable for approximately 48 months when stored protected from light at 5°C ± 3°C.

[0039] In some embodiments, the stability of the pharmaceutical composition disclosed herein is determined by size exclusion high-performance liquid chromatography (SE-HPLC). In one embodiment, the composition has a main peak (peak area %) of at least 95.0 as determined by SE-HPLC.

[0040] In some embodiments, the stability of the pharmaceutical composition disclosed herein is determined by imaging capillary isoelectric focusing (icIEF). In one embodiment, the composition has a main peak (peak area %) of at least 58.0, an acidic region (peak area %) of up to 32.0, and an alkaline region (peak area %) of up to 12.0, as determined by icIEF.

[0041] In some embodiments, the pharmaceutical compositions of this disclosure are reconstituted with sterile water for injection (SWFI). In some embodiments, the pharmaceutical composition is reconstituted in about 7.2 mL of SWFI. In some embodiments, the reconstituted composition is stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some embodiments, the reconstituted composition is stably stored at about 2°C to about 8°C for at least 24, 48, or 72 hours. In some embodiments, the reconstituted composition is further diluted in isotonic buffer in an intravenous (IV) infusion bag. In some embodiments, the final volume of the diluted composition in the IV infusion bag is between about 50 mL and about 100 mL. In some embodiments, the concentration of the immunoconjugate in the IV infusion bag is between about 0.72 mg and about 2.7 mg.

[0042] This article also provides a pharmaceutical composition prepared by lyophilization of a liquid formulation comprising 20 mg / mL of an anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v polysorbate 20, and 120 mM sucrose, wherein the liquid formulation has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0043]

[0044] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5 (such as about 3.5).

[0045] A liquid composition (such as for intravenous administration) is provided comprising: a) a concentration of polatuzumab vedotin between about 0.72 mg / mL and about 2.7 mg / mL; b) a concentration of sodium succinate between about 0.36 mM and about 1.35 mM; c) a concentration of sucrose between about 0.51 mM and about 16.24 mM; and d) a concentration of polysorbate 20 between about 0.0432 mg / mL and about 0.162 mg / mL, wherein the pH of the liquid composition is between about 5 and about 5.7. A liquid composition (such as for intravenous administration) is also provided, comprising: a) about 0.72 mg / mL polatuzumab vedotin; b) about 0.36 mM sodium succinate; c) about 0.51 mM sucrose; d) about 0.0432 mg / mL polysorbate 20, wherein the pH of the liquid composition is between 5.1 and about 5.4. A liquid composition (such as for intravenous administration) is also provided, comprising: a) about 2.7 mg / mL polatuzumab vedotin; b) about 1.35 mM sodium succinate; c) about 16.24 mM sucrose; d) about 0.162 mg / mL polysorbate 20, wherein the pH of the liquid composition is between about 5.1 and about 5.4. In some embodiments, the volume of the liquid composition is between about 50 mL and about 100 mL. In some embodiments, the volume of the liquid composition is 50 mL. In some embodiments, the volume of the liquid composition is 100 mL. In some embodiments, the liquid composition is contained in an intravenous (IV) infusion bag. In some embodiments, the surface of the IV infusion bag in contact with the liquid composition is composed of polyvinyl chloride (PVC), polyolefin (PO), polyethylene (PE), or polypropylene (PE).

[0046] In one aspect, this disclosure provides a pharmaceutical composition comprising an anti-CD79b immunoconjugate, a surfactant, a succinate buffer, and a sugar, wherein the pharmaceutical composition, when reconstituted in water, forms a liquid formulation comprising an anti-CD79b immunoconjugate at a concentration of about 10 mg / mL to about 20 mg / mL, a surfactant at a concentration of at least 0.06% w / v (i.e., 0.6 mg / mL), a succinate buffer at a concentration of about 10 mM to about 200 mM, and a sugar at a concentration of about 100 mM to about 260 mM, wherein the liquid formulation has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0047]

[0048] Ab is an anti-CD79b antibody, which comprises a heavy chain and a light chain. The light chain comprises: (a) the HVR-L1 sequence shown in an KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3). The heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6). Val is valine; Cit is citrulline; and p is from about 1 to about 8 (e.g., between about 2 and about 5). Between, such as about 3.5). In some embodiments, the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10.

[0049] In some embodiments, the pharmaceutical composition is reconstituted in SWFI and then diluted in a buffer solution in an IV infusion bag. In some embodiments, the pharmaceutical composition is reconstituted in SWFI and then diluted in an isotonic buffer solution in an IV infusion bag. In one embodiment, the concentration of the diluted surfactant in the IV infusion bag is at least 0.003% w / v. In one embodiment, the concentration of the diluted surfactant in the IV infusion bag is at least 0.004% w / v. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one specific embodiment, the concentration of sucrose is 120 mM. In some embodiments, the succinate buffer is sodium succinate buffer. In one specific embodiment, the concentration of sodium succinate buffer is 10 mM. In some embodiments, the pharmaceutical composition, after reconstitution, is stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some embodiments, the pharmaceutical composition, after reconstitution, can be stored stably at about 2°C to about 8°C for at least 24, 48, or 72 hours.

[0050] In some embodiments, the pharmaceutical composition, after being reconstituted in water, remains stable at 30°C for a maximum of about 1 day, a maximum of about 2 days, or a maximum of about 3 days. In some embodiments, the pharmaceutical composition, after being reconstituted in water, remains stable at 5°C ± 3°C for a maximum of about 1 day, a maximum of about 2 days, a maximum of about 3 days, a maximum of about 4 days, a maximum of about 5 days, a maximum of 6 days, or a maximum of about 7 days. In some embodiments, the stability of the composition is determined by size exclusion high-performance liquid chromatography (SE-HPLC). In one specific embodiment, the composition has a main peak (peak area %) of at least 95.0% as determined by SE-HPLC.

[0051] In some embodiments, the stability of the composition was measured by imaging capillary isoelectric focusing (icIEF). In one specific embodiment, the composition has a main peak of at least 58.0 (peak area %), an acidic region of up to 32.0 (peak area %), and an alkaline region of up to 12.0 (peak area %) as measured by icIEF.

[0052] In some embodiments, the pharmaceutical compositions disclosed herein are contained in glass vials (e.g., 20 mL glass vials). In some embodiments, the pharmaceutical compositions are lyophilized pharmaceutical compositions. In some embodiments, the pharmaceutical compositions are liquid pharmaceutical compositions.

[0053] In one aspect, this disclosure provides a pharmaceutical composition prepared by a method comprising the steps of: (a) lyophilizing a liquid composition to produce a lyophilized composition comprising 20 mg / mL of an anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v of polysorbate 20, and 120 mM sucrose, wherein the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0054]

[0055] Wherein: (a) Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5; (b) reconstitute the lyophilized composition with about 7.2 mL of sterile water for injection (SWFI) to produce a reconstituted composition; and (c) dilute the reconstituted composition in an isotonic buffer in an intravenous (IV) infusion bag to produce a pharmaceutical composition, wherein the final volume of the pharmaceutical composition in the IV infusion bag is about 100 mL, and wherein the final concentration of the immunoconjugate in the pharmaceutical composition is about 0.72 mg / mL or about 2.7 mg / mL.

[0056] In one aspect, this disclosure provides a liquid composition comprising an anti-CD79b immunoconjugate, a surfactant, a succinate buffer, and a sugar, wherein the concentration of the anti-CD79b immunoconjugate is from about 10 mg / mL to about 20 mg / mL, the concentration of the surfactant is at least 0.06% w / v (i.e., 0.6 mg / mL), the concentration of the succinate buffer is from about 10 mM to about 200 mM, and the concentration of the sugar is from about 100 mM to about 260 mM, wherein the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0057]

[0058] in:

[0059] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (e.g., between about 2 and about 5). Between, such as about 3.5). In some embodiments, the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10.

[0060] In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one specific embodiment, the concentration of sucrose is 120 mM. In some embodiments, the succinate buffer is sodium succinate buffer. In one specific embodiment, the concentration of sodium succinate buffer is 10 mM.

[0061] In some embodiments, the liquid composition of this disclosure is diluted in an isotonic buffer. In one exemplary embodiment, the liquid composition of this disclosure is dissolved in an isotonic buffer in an IV infusion bag. In some embodiments, the isotonic buffer used to dilute the liquid composition is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, or a 5% glucose solution.

[0062] In some embodiments, the liquid compositions provided herein, when diluted in a 0.9% sodium chloride solution, remain stable at 2°C–8°C for a maximum of at least about 24 hours (according to any one or more criteria described elsewhere herein). In some embodiments, the liquid compositions provided herein, when diluted in a 0.9% sodium chloride solution, remain stable at 9°C to 25°C for a maximum of at least 4 hours (according to any one or more criteria described elsewhere herein). In some embodiments, the liquid compositions provided herein, when diluted in a 0.45% sodium chloride solution, remain stable at 2°C–8°C for a maximum of at least about 24 hours (according to any one or more criteria described elsewhere herein). In some embodiments, the liquid compositions provided herein, when diluted in a 0.45% sodium chloride solution, remain stable at 9°C to 25°C for a maximum of at least about 4 hours (according to any one or more criteria described elsewhere herein). In some embodiments, the liquid compositions provided herein, when diluted in a 5% glucose solution, remain stable at 2°C–8°C for a maximum of at least about 48 hours (according to any one or more criteria described elsewhere herein). In some embodiments, the liquid compositions provided herein, when diluted in a 5% glucose solution, remain stable at 9°C to 25°C for a maximum of at least about 8 hours (according to any one or more criteria described elsewhere herein).

[0063] In some embodiments, the liquid composition diluted in isotonic buffer is contained in an IV infusion bag. In some embodiments, the surface of the IV infusion bag in contact with the composition diluted in isotonic buffer is composed of polyvinyl chloride (PVC), polyolefin (PO), polyethylene (PE), or polypropylene (PE).

[0064] In some embodiments, the liquid composition of this disclosure diluted in an isotonic buffer solution in an IV infusion bag remains stable at 30°C for up to about 6 to about 8 hours. In some embodiments, the liquid composition of this disclosure dissolved in an isotonic buffer solution in an IV infusion bag remains stable at 25°C for up to about 24 hours. In some embodiments, the liquid composition of this disclosure dissolved in an isotonic buffer solution in an IV infusion bag remains stable at 5°C ± 3°C for up to about 72 hours. In some embodiments, the isotonic buffer solution is physiological saline. In some embodiments, the liquid composition is stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some embodiments, the liquid composition is stably stored at about 2°C to about 8°C for at least 24, 48, or 72 hours.

[0065] A lyophilized pharmaceutical composition is also provided, comprising about 150 mg of an anti-CD79b immunoconjugate, about 9.0 mg of polysorbate 20, about 8.88 mg of succinic acid, about 4.08 mg of sodium hydroxide, and about 309 mg of sucrose, wherein the anti-CD79b immunoconjugate comprises the formula:

[0066]

[0067] Wherein, Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5 (such as about 3.5). In some embodiments, the lyophilized composition (such as a block) is contained in a bottle (e.g., a 20 mL glass bottle).

[0068] A further lyophilized pharmaceutical composition is provided, comprising about 150 mg of polatuzumab vedotin, about 9.0 mg of polysorbate 20, about 8.88 mg of succinic acid, about 4.08 mg of sodium hydroxide, and about 309 mg of sucrose. In some embodiments, the lyophilized composition (such as a block) is contained in a bottle (e.g., a 20 mL glass bottle).

[0069] A lyophilized pharmaceutical composition is also provided, comprising approximately 140 mg of an anti-CD79b immunoconjugate, approximately 8.4 mg of polysorbate 20, approximately 8.27 mg of succinic acid, approximately 3.80 mg of sodium hydroxide, and approximately 288 mg of sucrose, wherein the anti-CD79b immunoconjugate comprises the formula:

[0070]

[0071] Wherein b is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5 (such as about 3.5). In some embodiments, the lyophilized composition (such as a block) is contained in a bottle (e.g., a 20 mL glass bottle).

[0072] A further lyophilized pharmaceutical composition is provided, comprising about 140 mg of polatuzumab vedotin, about 8.4 mg of polysorbate 20, about 8.27 mg of succinic acid, about 3.80 mg of sodium hydroxide, and about 288 mg of sucrose. In some embodiments, the lyophilized composition (such as a block) is contained in a bottle (e.g., a 20 mL glass bottle).

[0073] A lyophilized pharmaceutical composition is also provided, comprising about 30 mg of an anti-CD79b immunoconjugate, about 1.8 mg of polysorbate 20, about 1.77 mg of succinic acid, about 0.816 mg of sodium hydroxide, and about 61.8 mg of sucrose, wherein the anti-CD79b immunoconjugate comprises the formula:

[0074]

[0075] Wherein, Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5 (such as about 3.5). In some embodiments, the lyophilized composition is a lyophilized block. In some embodiments, the lyophilized composition (such as a block) is contained in a bottle (e.g., a 20 mL glass bottle).

[0076] A further lyophilized pharmaceutical composition is provided, comprising about 30 mg of polatuzumab vedotin, about 1.8 mg of polysorbate 20, about 1.77 mg of succinic acid, about 0.816 mg of sodium hydroxide, and about 61.8 mg of sucrose. In some embodiments, the lyophilized composition (such as a block) is contained in a bottle (e.g., a 20 mL glass bottle).

[0077] In some embodiments, the lyophilized pharmaceutical compositions provided herein are lyophilized blocks. In some embodiments, the lyophilized pharmaceutical compositions according to any one of the embodiments described herein can be stably stored at about 2°C to about 8°C for at least 6, 12, 18, 24, 30, 36, 42, 48, 54 or 60 months.

[0078] A liquid pharmaceutical composition is provided comprising: a) a concentration of about 5-60 mg / mL of polatuzumab vedotin; b) a concentration of about 10-200 mM of sodium succinate; c) a concentration of about 100-260 mM of sucrose; and d) a concentration of about 0.06-0.12% w / v of polysorbate 20, wherein the pH of the liquid composition is between 5 and 6. In some embodiments, the liquid pharmaceutical composition comprises: a) a concentration of about 10-55 mg / mL of polatuzumab vedotin; b) a concentration of about 10-100 mM of sodium succinate; c) a concentration of about 150-260 mM of sucrose; and d) a concentration of about 0.08-0.12% w / v of polysorbate 20, wherein the pH of the liquid composition is between 5.1 and 5.6. In some embodiments, the liquid pharmaceutical composition comprises: a) polatuzumab vedotin at a concentration of about 15-40 mg / mL; b) sodium succinate at a concentration of about 10-50 mM; c) sucrose at a concentration of about 200-260 mM; and d) polysorbate 20 at a concentration of about 0.1-0.12% w / v, wherein the pH of the liquid composition is between 5.2 and 5.4. In some embodiments, the liquid composition is obtained by reconstitution of a lyophilized composition (such as a block). In some embodiments, the liquid composition is contained in a bottle (e.g., a 20 mL glass bottle).

[0079] A liquid pharmaceutical composition is also provided, comprising: a) 20 mg / mL polatuzumab vedotin; b) 10 mM sodium succinate; c) 120 mM sucrose; and d) 0.12% w / v polysorbate 20, wherein the pH of the liquid composition is about 5.3. In some embodiments, the liquid composition is obtained by reconstitution of a lyophilized composition (such as a block). In some embodiments, the liquid composition is contained in a bottle (e.g., a 20 mL glass bottle).

[0080] In some embodiments, the liquid composition according to any one of the examples described herein is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some embodiments, the liquid composition according to any one of the examples described herein is stably stored at about 2°C to about 8°C for at least about 24, 48, or 72 hours. In some embodiments, the liquid composition is obtained by reconstituted lyophilized composition (such as a block). In some embodiments, the liquid composition is contained in a bottle (e.g., a 20 mL glass bottle).

[0081] In some embodiments, the reconstituted pharmaceutical compositions provided herein remain stable after storage at 2°C–8°C for 72 hours. In some embodiments, the reconstituted pharmaceutical compositions provided herein remain stable after storage at 30°C for 24 hours after exposure to ambient light. In some embodiments, the reconstituted pharmaceutical compositions comprise 20 mg / mL polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, and 1.2 mg / mL polysorbate 20, wherein the pH is 5.3. In some embodiments, the affinity of polatuzumab vedotin for its target (i.e., CD79b) or the bioactivity of polatuzumab vedotin is discussed.

[0082] In one aspect, this disclosure provides a method of treating a proliferative disorder in a patient in need, the method comprising administering to the patient a pharmaceutical composition or liquid composition described herein. Use of the pharmaceutical composition or liquid composition described herein in the production of a medicament for treating a proliferative disorder in a patient in need is also provided. In some embodiments, use of the pharmaceutical composition or liquid composition described herein in treating a proliferative disorder in a patient in need is provided. Use of the pharmaceutical composition or liquid composition described herein in a method for treating a proliferative disorder in a patient in need is also provided.

[0083] In some embodiments, the proliferative disorder is cancer. In one exemplary embodiment, the cancer is a B-cell proliferative disorder. In a specific embodiment, the B-cell proliferative disorder is selected from the group consisting of: lymphoma, myeloma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), relapsed / refractory DLBCL, aggressive NHL, indolent lymphoma, follicular lymphoma (FL), relapsed aggressive NHL, relapsed indolent NHL, relapsed NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma.

[0084] In one embodiment of this disclosure, the B-cell proliferative disorder is non-Hodgkin lymphoma (NHL). In one embodiment, the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL). In one embodiment, the B-cell proliferative disorder is relapsed / refractory DLBCL. In some embodiments, the B-cell proliferative disorder is relapsed / refractory DLBCL. In another embodiment, the B-cell proliferative disorder is relapsed NHL or refractory NHL. In yet another embodiment, the B-cell proliferative disorder is follicular lymphoma (FL). Attached Figure Description

[0085] Figure 1 shows the increase in high molecular weight substances (HMWS) after static storage at 30°C for 22 hours in three different sizes of intravenous (IV) infusion bags containing a fixed concentration of PS20 ([PS20]).

[0086] Figure 2 shows the increase in HMWS after stirring at 30°C (100 rpm) for 2 hours in three different sizes of IV infusion bags containing a fixed concentration of PS20 ([PS20]).

[0087] Figure 3 shows the effect of temperature on the physical stability of anti-CD79b-vc-MMAE in saline IV infusion bags containing different concentrations of PS20 ([PS20]).

[0088] Figure 4. Effects of surfactant and infusion type on static storage at 30°C for 22 hours.

[0089] Figure 5A shows the effect of temperature (2-8℃) on the stability of resistance to CD79b-vc-MMAE under stirring. Figure 5B shows the effect of temperature (25℃) on the stability of resistance to CD79b-vc-MMAE under stirring. Figure 5C shows the effect of temperature (30℃) on the stability of resistance to CD79b-vc-MMAE under stirring.

[0090] Figure 6 shows the physical stability against CD79b-vc-MMAE obtained using stirring model 1.

[0091] Figure 7 shows the physical stability against CD79b-vc-MMAE obtained using stirring model 2.

[0092] Figure 8 illustrates the hydrolysis reaction of thiosuccinimide used to stabilize the immunoconjugate and prevent its elimination. Hydrolysis of the thiosuccinimide bond present on the immunoconjugate prevents maleimide elimination.

[0093] Figure 9 shows the effect of pH on the formation of acidic charge variants at 30°C at three different time points: 2 weeks, 4 weeks, and 8 weeks.

[0094] Figure 10 shows the effect of pH on the formation of basic charge variants at 30°C at three different time points: 2 weeks, 4 weeks, and 8 weeks.

[0095] Figure 11 shows the effect of pH on HMWS formation at 30°C at three different time points: 2 weeks, 4 weeks, and 8 weeks.

[0096] Figure 12 shows the effect of pH and buffer type on low molecular weight substances (LMWS) at 30°C for 4 weeks.

[0097] Figure 13 shows the stability of the lyophilized formulation obtained by size exclusion chromatography (SEC) at 30°C.

[0098] Figure 14 shows the stability of the lyophilized formulation obtained by imaging capillary isoelectric focusing (icIEF) at 30°C.

[0099] Figures 15A to 15C show the appearance of freeze-dried blocks with different protein-to-sucrose ratios. Figure 15A shows the results of 10 mg / mL anti-CD79b-vc-MMAE with 260 mM sucrose. Figure 15B shows the results of 10 mg / mL anti-CD79b-vc-MMAE with 180 mM sucrose. Figure 15C shows the results of 10 mg / mL anti-CD79b-vc-MMAE with 120 mM sucrose.

[0100] Figures 16A and 16B show the appearance of freeze-dried blocks with different protein-to-sucrose ratios. Figure 16A shows the results of 10 mg / mL anti-CD79b-vc-MMAE with 260 mM sucrose. Figure 16B shows the results of 20 mg / mL anti-CD79b-vc-MMAE with 120 mM sucrose.

[0101] Figure 17 shows the stability of the lyophilized anti-CD79b-vc-MMAE drug at 2-8°C, 25°C, and 40°C, where the HMWS variant content was determined by SEC.

[0102] Figure 18 shows the stability of the lyophilized anti-CD79b-vc-MMAE drug at 2-8°C, 25°C, and 40°C, where the HMWS variant content was determined by icIEF. Detailed Implementation

[0103] This disclosure provides a stable pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant. This disclosure also provides a method of treating cancer using such a composition.

[0104] I. Definition

[0105] It should be understood that this disclosure is not limited to specific compositions or biological systems, which can certainly vary. Furthermore, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” “the,” and “described” include plural referents unless the context clearly specifies otherwise. Thus, for example, a reference to “molecule” includes a combination of two or more such molecules, etc.

[0106] As used herein, the term "about" refers to a common range of error for the corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments relating to that value or parameter itself.

[0107] It should be understood that the aspects and embodiments disclosed herein include those referred to as “comprising,” “consisting of,” and “substantially consisting of.”

[0108] The term "anti-CD79b immunoconjugate" refers to an anti-CD79b antibody-drug conjugate (ADC). As used herein, an anti-CD79b immunoconjugate comprises an antibody or fragment thereof capable of binding to CD79b, a linker, and a drug molecule. The term "linker" is used to refer to 6-maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB).

[0109] The term “antibody” is used in the broadest sense and specifically includes, for example, single anti-CD79b monoclonal antibodies (including, but not limited to, agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), anti-CD79b antibody compositions with multi-epitope specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), single-chain anti-CD79b antibodies, and fragments of anti-CD79b antibodies (see below, including Fab, Fab', F(ab')2, and Fv fragments), bisomal antibodies, and single-domain antibodies (sdAbs), provided they exhibit the desired biological or immunomodulatory activity. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein. Antibodies can be chimeric antibodies, human antibodies, humanized antibodies, and / or affinity-matured antibodies.

[0110] The terms "anti-CD79b antibody" and "CD79b-binding antibody" refer to antibodies that bind to CD79b with sufficient affinity, making them usable as diagnostic and / or therapeutic agents targeting CD79b. Preferably, as measured, for example by radioimmunoassay (RIA), the anti-CD79b antibody binds to unrelated non-CD79b proteins to less than about 10% of its binding to CD79b. In some embodiments, the CD79b-binding antibody has a dissociation constant (Kd) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In some embodiments, the anti-CD79b antibody binds to an epitope of CD79b that is conserved across different species of CD79b.

[0111] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies consist of 5 basic heterotetrameric units and a polypeptide called the J chain, thus containing 10 antigen-binding sites, while secretory IgA antibodies can polymerize to form a multivalent combination containing 2-5 basic 4-chain units and the J chain). In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to the H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isoform of the H chain. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) (for each α and γ chain) and four CH domains (for μ and ε isoforms). Each L chain has a variable domain (VL) at the N-terminus and a constant domain (CL) at the other end. The VL pairs with the VH, and the CL pairs with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form interfaces between the variable domains of the light and heavy chains. Paired VH and VL together form a single antigen-binding site. For information on the structure and properties of different classes of antibodies, see, for example: *Basic and Clinical Immunology*, 8th ed., Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0112] L-chains derived from any vertebrate, based on the amino acid sequence of their constant domain, can be classified into one of two distinct types, referred to as Kappa (κ) and Lambda (λ). Immunoglobulins, based on the amino acid sequence of their heavy chain constant domain (CH), can be classified into different classes or isotypes. Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, with heavy chains referred to as α, δ, ε, γ, and μ, respectively. The γ and α classes are further subdivided based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0113] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain is called "VH," and the variable domain of the light chain is called "VL." These domains are typically the most variable parts of an antibody and contain antigen-binding sites.

[0114] The term "variable" refers to the significant differences in certain segments of the variable domain between antibody sequences. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, the variability is not uniformly distributed among the 110 amino acids of the variable domain. Instead, the V region consists of relatively constant frames (FRs) of 15–30 amino acids, separated by much more variable shorter regions called "hypervariates," each 9–12 amino acids long. The variable domains of the natural heavy and light chains each contain four FRs, primarily employing a β-sheet structure, connected by three hypervariable regions that form a loop connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. Hypervariable regions in each chain are tightly bound together by FRs and, together with hypervariable regions in another chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD (1991)). Constant domains do not directly participate in antibody-antigen binding but have their own effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC).

[0115] An "intact" antibody is an antibody that contains an antigen-binding site, a CL, and at least heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, an intact antibody has one or more effector functions.

[0116] For the purposes of this article, "naked antibody" refers to an antibody that is not conjugated to a drug component or a radiolabel.

[0117] An "antibody fragment" comprises a portion of a complete antibody, preferably containing the antigen-binding or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bisomatic antibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Engineering 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment contains the antigen-binding site of the complete antibody, thus preserving the ability to bind antigens.

[0118] Papain digestion of antibodies produces two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment (the name reflects its tendency to crystallize). The Fab fragment consists of the complete L chain, a variable region domain (VH) of the H chain, and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in terms of antigen binding; that is, it has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, exhibiting divalent antigen-binding activity and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment in that the Fab' fragment has residues added to the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the designation used herein for Fab' fragments in which the cysteine ​​residues of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with a hinge cysteine ​​residue in between. Other chemical couplings of antibody fragments are also known.

[0119] The Fc fragment contains the carboxyl-terminal portion of two H chains linked together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also the part recognized by Fc receptors (FcRs) present on certain types of cells.

[0120] An “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a tightly bound, non-covalently linked dimer of a heavy chain variable region domain and a light chain variable region domain. In the single-chain Fv (scFv) species, a heavy chain variable region domain and a light chain variable region domain can be covalently linked via a flexible peptide linker, allowing the light and heavy chains to associate into a “dimer” structure similar to that in the double-chain Fv species. The folding of these two domains produces six hypervariable rings (three rings each from the H and L chains), which contribute amino acid residues to achieve antigen binding, thus giving the antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, although with lower affinity than a complete binding site.

[0121] A “single-chain Fv”, also abbreviated as “sFv” or “scFv”, is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the sFv to form the desired antigen-binding structure. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994); Borrebaeck 1995, hereinafter.

[0122] The term "bimeric antibody" refers to an antibody fragment with two antigen-binding sites, containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). Small antibody fragments are prepared by constructing sFv fragments (see previous section) with short linkers (approximately 5-10 residues) between the VH and VL domains, thereby achieving interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bimeric antibodies can be either bivalent or bispecific. Bispecific bimeric antibodies are heterodimers of two "crossed" sFv fragments, where the VH and VL domains of the two antibodies are located on different polypeptide chains. Bivalent antibodies are described more comprehensively in, for example, EP 404,097; WO 93 / 11161; Hudson et al., *Nature Medicine* 9: 129-134 (2003); and Hollinger et al., *Proceedings of the National Academy of Sciences of the United States of America* 90: 6444-6448 (1993). Hudson et al. also described trisomic and tetrasomic antibodies in *Nature Medicine* 9: 129-134 (2003).

[0123] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies in the group are identical except for a small number of naturally occurring mutations that may be present. Monoclonal antibodies exhibit high specificity for a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. For example, it can be prepared using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or it can be prepared in bacterial, eukaryotic, or plant cells using recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, those described by Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., Journal of Molecular Biology, 222: 581-597 (1991).

[0124] The monoclonal antibodies described in this article include “chimeric” antibodies, in which a portion of the heavy and / or light chains is identical or homologous to the corresponding sequences in antibodies from a specific species or belonging to a specific antibody class or subclass, and the remainder of one or more chains is identical or homologous to the corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of those antibodies, provided they exhibit the desired biological activity (see US No. 4,816,567; and Morrison et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 81:6851-6855 (1984)). The target chimeric antibodies described in this article include “primate” antibodies, which contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0125] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing a minimal sequence derived from a non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), where residues in the hypervariable region of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates, which possess the desired antibody specificity, affinity, and function. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not present in the receptor antibody or donor antibody. These modifications are intended to further refine antibody performance. Generally, humanized antibodies will substantially contain at least one variable domain, typically two, where all or substantially all hypervariable loops correspond to hypervariable loops of the non-human immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), which is typically a human immunoglobulin. For more details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See also the following review articles and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1: 105-115 (1998); Harris, Biochem. Soc. Transactions, 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5: 428-433 (1994).

[0126] "Human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody prepared using any of the techniques disclosed herein for the preparation of human antibodies. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Methods also available for the preparation of human monoclonal antibodies are described in the following literature: Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1): 86-95 (1991). See also van Dijk and van de Winkel, *New Insights in Pharmacology*, 5: 368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been deactivated, for example, immunized xenogeneic mice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 relating to XENOMOUSE™ technology). See also, for example, Li et al., *Proceedings of the National Academy of Sciences of the United States of America*, 103: 3557-3562 (2006) on human antibodies produced via human B-cell hybridoma technology.

[0127] As used herein, the terms “hypervariant region,” “HVR,” or “HV” refer to regions of antibody variable domains that are hypervariable in sequence and / or form structurally defined loops. Typically, antibodies contain six hypervariant regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Many descriptions of hypervariant regions are used and are included herein. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD. (1991)). Conversely, the Chothia term refers to the location of a structural loop (Chothia and Lesk, *Journal of Molecular Biology*, 196: 901-917 (1987)). When numbered using the Kabat numbering scheme, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertion points at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between the Kabat CDR and Chothia structural loops and are used by the AbM antibody modeling software from Oxford Molecular. The “contact” hypervariable regions are based on analysis of available complex crystal structures. Each residue in these hypervariable regions is described below.

[0128]

[0129] The hypervariable region may include the following “extended hypervariable regions”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 (L3) in VL, and 26-35B (H1), 50-65, 47-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to the method described above by Kabat et al.

[0130] "Frame" or "FR" residues are those variable domain residues other than the hypervariable region residues defined in this paper.

[0131] The terms “Kabat-described variable domain residue numbering” or “Kabat-described amino acid position numbering” and their variations refer to the numbering system proposed by Kabat et al. for heavy chain or light chain variable domains in the following literature: *Sequences of Proteins of Immunological Interest*, 5th edition, U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortened or inserted FR or CDRs of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat numbering) after residue 52 of H2 and insertion residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat numbering). The Kabat number of a given antibody residue can be determined by comparing the antibody sequence with homologous regions of a “standard” Kabat-numbered sequence.

[0132] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported by Kabat et al. above). “The EU index described by Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to residue numbers obtained through the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbers obtained through the EU numbering system (e.g., see U.S. Provisional Application No. 60 / 640,323, EU numbering).

[0133] Affinity-matured antibodies are antibodies in which one or more HVR domains undergo one or more alterations, resulting in improved affinity of the antibody for the antigen compared to the parent antibody without these alterations. Preferred affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced by methods known in the art. Marks et al. in *Bio / Technology* 10:779-783 (1992) described affinity maturation via VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues is described in the following literature: Barbas et al., Proc. Natl. Acad. Sci. USA 91: 3809-3813 (1994); Schier et al., Gene 169: 147-155 (1995); Yelton et al., Journal of Immunology 155: 1994-2004 (1995); Jackson et al., Journal of Immunology 154(7): 3310-9 (1995); and Hawkins et al., Journal of Molecular Biology 226: 889-896 (1992).

[0134] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind antigens rapidly and tend to maintain binding for longer periods. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific exemplary embodiments are described below.

[0135] The term "or better" binding affinity as used in this article refers to a stronger binding force between the molecule and its binding partner. As used herein, "or better" means a stronger binding force, expressed as a smaller Kd value. For example, when an antibody's affinity for an antigen is mentioned as ".6 nM or better," the antibody's affinity for the antigen is < .6 nM, i.e., .59 nM, .58 nM, .57 nM, or any value less than .6 nM.

[0136] In one embodiment, the “Kd” or “Kd value” according to this disclosure is measured by a radiolabeled antigen binding assay (RIA) performed as described below using a target antibody and its antigen in Fab form, the assay measuring the solution-binding affinity of the antigen to Fab by equilibrating Fab with a minimum concentration of (125I)-labeled antigen in the presence of a series of titrations of unlabeled antigen, followed by capture of the bound antigen with a plate coated with anti-Fab antibody (Chen et al., (1999) J. Mol Biol 293: 865-881). To determine the conditions used for the assay, a microtiter plate (Dynex) was coated overnight with 50 mM sodium carbonate (pH 9.6) containing 5 μg / mL capture anti-Fab antibody (Cappel Labs), and subsequently blocked for two to five hours in PBS containing 2% (w / v) bovine serum albumin at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125I]-antigen was mixed with serial dilutions of the Fab of interest (e.g., following the evaluation of anti-VEGF antibody (Fab-12) in Presta et al., (1997) Cancer Res. 57: 4593-4599). The Fab of interest was then incubated overnight; however, incubation could be prolonged (e.g., 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution was then removed and the plate was washed eight times with PBS containing 0.1% Tween-20. When the plate was dry, 150 μl / well of scintillator (MicroScint-20; Packard) was added, and the plate was counted for several tens of minutes on a TOPCOUNT™ γ counter (Packard). Select the concentration of each Fab that has a maximum binding rate of less than or equal to 20% for use in competitive binding assays.

[0137] According to another embodiment, at 25°C, using an immobilized antigen CM5 chip, the “Kd” or “Kd value” was measured by surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 10 response units (RU). In short, the carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. The antigen was diluted to 5 μg / mL (approximately 0.2 µM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 µL / min to obtain approximately 10 response units (RU) of conjugate protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25 °C, Fab was injected in serially diluted (0.78 nM to 500 nM) two-fold dilutions in PBS (PBST) containing 0.05% Tween 20 at a flow rate of approximately 25 µL / min. The association rate (k-value) was calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting association and dissociation sensor maps. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, for example, Chen, Y. et al., (1999) *Journal of Molecular Biology* (J. Mol Biol) 293:865-881. If the association rate obtained by the above surface plasmon resonance determination exceeds 10... 6 M -1 S -1 The association rate can be determined using fluorescence quenching techniques, i.e., by measuring the increase or decrease of fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) in PBS (pH 7.2) containing 20 nM anti-antigen antibody (Fab form) at 25 °C in the presence of gradually increasing antigen concentrations in a spectrometer such as an Aviv Instruments spectrophotometer equipped with a flow stop device or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0138] According to the "association rate" or "rate of association" or "k" as described in this disclosure on "Measurements can also be performed using the same surface plasmon resonance technique described above, using the BIAcore™-2000 or BIAcore™-3000 instruments (BIAcore, Inc., Piscataway, NJ) as described above."

[0139] As used herein, the phrase "substantially similar" or "substantially identical" means that there is a sufficiently high degree of similarity between two values ​​(typically one value is associated with the antibody described in this disclosure, and the other with a reference / control antibody), thereby leading one skilled in the art to believe that the difference between the two values ​​has little biological and / or statistical significance in the context of the biological characteristic measured by the values ​​(e.g., Kd values). Depending on the value of the reference / control antibody, the difference between the two values ​​is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, and preferably less than about 10%.

[0140] As used herein, the phrases “significantly reduced” or “significantly different” indicate a sufficiently high difference between two values ​​(typically one value associated with the antibody described in this disclosure and the other with a reference / control antibody), such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the values ​​(e.g., Kd value, HAMA response). Depending on the value of the reference / control antibody, the difference between the two values ​​is preferably greater than about 10%, preferably greater than about 20%, preferably greater than about 30%, preferably greater than about 40%, and preferably greater than about 50%.

[0141] For the purposes of this document, a “recipient human frame” is a frame containing an amino acid sequence of a VL or VH frame derived from a human immunoglobulin frame or a human common frame. A recipient human frame “derived from” a human immunoglobulin frame or a human common frame may contain the same amino acid sequence as said human immunoglobulin frame or human common frame, or may contain pre-existing amino acid sequence variations. Where pre-existing amino acid variations are present, preferably no more than five pre-existing amino acid variations are present, and preferably four or fewer, or three or fewer pre-existing amino acid variations are present. Where pre-existing amino acid variations are present in VH, preferably those variations are only at three, two, or one of positions 71H, 73H, and 78H; for example, the amino acid residues at those positions may be 71A, 73T, and / or 78A. In one embodiment, the VL recipient human frame is sequence-identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

[0142] A “human common framework” is a framework that represents the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, the sequence subgroup is the subgroup described by Kabat et al. In one embodiment, for VL, the subgroup is the κ I subgroup described by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III described by Kabat et al.

[0143] The “VH Subgroup III Common Framework” comprises a common sequence obtained from the amino acid sequence of the variable weight subgroup III as described by Kabat et al. In one embodiment, the VH Subgroup III Common Framework amino acid sequence comprises at least a portion or all of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 11)-H1-WVRQAPGKGLEWV (SEQ ID NO: 12)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 145)-H3-WGQGTLVTVSS (SEQ ID NO: 13).

[0144] The “VL Subgroup I Common Framework” comprises a common sequence obtained from the amino acid sequence of the variable light κ subgroup I as described by Kabat et al. In one embodiment, the VL Subgroup I Common Framework amino acid sequence comprises at least some or all of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 14)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 15)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 141)-L3-FGQGTKVEIKR (SEQ ID NO: 16).

[0145] "Unmodified human frames" are human frames that have the same amino acid sequence as the recipient human frames, for example, those that lack the substitution of non-human amino acids with human amino acids in the recipient human frames.

[0146] Anti-CD79b immunoconjugates, or antibodies that "bind" to CD79b, are antibodies that bind to the CD79b antigen with sufficiently high affinity, thereby enabling the antibody to be used as a therapeutic agent targeting cells or tissues expressing the antigen, and without significant cross-reactivity with other proteins. In such embodiments, the degree of binding of the antibody to "non-target" proteins is less than about 10% of the degree of binding of the antibody to its specific target protein, as measured by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). Regarding the binding of antibodies to target molecules, the terms "specific binding" or "specifically bound to" or "specific to" a specific polypeptide or epitope on a specific polypeptide target mean a distinct difference from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is typically a molecule with a similar structure but without binding activity. For example, specific binding can be measured by competition with a control molecule similar to the target (an excess of the unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of the unlabeled target, specific binding is indicated. As used herein, the terms "specific binding," "specific binding to," or "specific to" a specific peptide or an epitope on a specific peptide target can be, for example, having at least about 10 [units of something] on the target. -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11M, or at least about 10 -12 Molecules with an M or larger Kd. In one embodiment, the term "specific binding" refers to the binding of an antibody to an epitope on the CD79b peptide while substantially not binding to any other peptide or peptide epitope.

[0147] Antibody "effector function" refers to the biological activity attributed to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant), and varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0148] The term "Fc region" used herein is used to define the C-terminal region of the immunoglobulin heavy chain, which includes both the native sequence Fc region and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification or by recombinantly designing the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies may include antibody populations with all K447 residues removed, antibody populations with K447 residues not removed, and antibody populations having mixtures containing and without K447 residues.

[0149] A “functional Fc region” possesses the “effective function” of the native Fc region. Exemplary “effective functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the Fc region in combination with a binding domain (e.g., antibody variable domain) and can be assessed using various assays, such as those disclosed in the definitions herein.

[0150] The “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region and its naturally occurring variants.

[0151] The “variant Fc region” comprises an amino acid sequence different from the natural Fc region, achieved by means of at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the natural Fc region or the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions in the natural Fc region or the Fc region of the parent polypeptide, and preferably about one to about five amino acid substitutions. The variant Fc region described herein preferably has at least about 80% homology with the natural Fc region and / or with the Fc region of the parent polypeptide, and most preferably at least about 90% homology, more preferably at least about 95% homology.

[0152] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens, subsequently killing the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are essential for this type of killing. The main cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet's *Annu. Rev. Immunol.* 9:457-92 (1991). To assess the ADCC activity of a target molecule, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Optionally or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al., (USA) 95:652-656 (1998).

[0153] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are FcRs that bind to IgG antibodies (a type of γ-receptor) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. (See review: M. in Daëron, *Annu. Rev. Immunol.* 15: 203-234 (1997).) Reviews on FcRs can be found in: Ravetch and Kine, *Annu. Rev. Immunol.* 9: 457-492 (1991); Capel et al., *Immunomethods* 4: 25-34 (1994); and de Haas et al., *J. Lab. Clin. Med.* 126: 330-41 (1995). The term "FcR" as used in this article encompasses other FcRs, including those awaiting future identification. The term also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); and Kim et al., J. Immunol. 24: 249 (1994)).

[0154] In vivo binding to human FcRn and serum half-life of high-affinity human FcRn binding peptides can be determined, for example, in transgenic mice expressing human FcRn or in transfected human cell lines, or in primates given peptides with variant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0155] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, these cells express at least FcγRIII and perform ADCC effector functions. Human leukocytes mediating ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among which PBMCs and NK cells are preferred. Effector cells can be isolated from natural sources (e.g., blood).

[0156] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to an antibody (appropriate subclass) that binds to its homologous antigen. To assess complement activation, a CDC assay can be performed, as described in: Gazzano-Santoro et al., *Journal of Immunology* 202: 163 (1996). Peptide variants with altered Fc region amino acid sequences (peptides with variant Fc regions) and increased or decreased C1q binding capacity are described, for example, in U.S. Patent Nos. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al., *Journal of Immunology* 164: 4178-4184 (2000).

[0157] The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody purification or by recombinantly designing the nucleic acid encoding the antibody. Therefore, compositions comprising antibodies having an Fc region according to this disclosure may comprise antibodies having K447, antibodies with all K447 removed, or mixtures of antibodies containing and not containing K447 residues.

[0158] The “B cell surface markers” or “B cell surface antigens” described herein are antigens expressed on the surface of B cells that can be targeted by antagonists that bind to them, including but not limited to antibodies against B cell surface antigens or soluble forms of B cell surface antigens that can antagonize the binding of ligands to naturally occurring B cell antigens. Exemplary B cell surface markers include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, and CD86. (For a description, see *The Leukocyte Antigen Facts Book*, 2nd edition, 1997, edited by Barclay et al., Academic Press, HarcourtBrace & Co., New York). Other B cell surface markers include RP105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. These B cell surface markers of particular interest are preferably expressed on B cells, and can be expressed on both progenitor and mature B cells, compared to other non-B cell tissues in mammals.

[0159] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, cancers of the hematopoietic system or blood-related cancers such as lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as spleen cancer and lymph node cancer, and malignant tumors, blastomas, and sarcomas. More specific examples of cancer include B-cell-related cancers, such as high, intermediate, and low-grade lymphomas (including B-cell lymphomas, such as mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin lymphoma (NHL), mantle cell lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and Hodgkin lymphoma and T-cell lymphoma), as well as leukemias (including secondary leukemia, chronic lymphocytic leukemia (CLL) such as B-cell leukemia (CD5+ B lymphocytes), myeloid leukemia such as acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia such as allergic lymphocytic leukemia (ALL), and spinal dysplasia), and other cancers related to blood and / or B-cells or T-cells. This also includes cancers of other hematopoietic cells, including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils, and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells. It also includes cancerous B-cell proliferative disorders selected from the following: lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed-invasive NHL, relapsed-indolent NHL, refractory NHL, refractory-indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), DLBCL, relapsed / refractory DLBCL, FL, and mantle cell lymphoma. The origins of B-cell carcinomas include: marginal zone B-cell lymphoma originating from marginal B cells in the memory zone; follicular lymphoma and diffuse large B-cell lymphoma originating from centroblasts in the bright zone of the germinal center; chronic lymphocytic leukemia and small lymphocytic leukemia originating from B1 cells (CD5+); mantle cell lymphoma originating from primary B cells in the mantle layer; and Burkitt lymphoma originating from centroblasts in the dark zone of the germinal center. Tissues containing hematopoietic cells are referred to herein as "hematopoietic tissues," which include the thymus and bone marrow, as well as peripheral lymphoid tissues such as the spleen, lymph nodes, mucosa-associated lymphoid tissues such as intestinal lymphoid tissues, tonsils, Peyer's cluster lymph nodes, and other mucosa-associated appendix and lymphoid tissues such as the endobronchial lining.More specific examples of this type of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancer.

[0160] The terms "B-cell malignancies" or "B-cell proliferative disorders" used in this article include non-Hodgkin lymphoma (NHL), including low-grade / follicular NHL, small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade non-cleavage small cell NHL, giant cell NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom macroglobulinemia, non-Hodgkin lymphoma (NHL), DLBCL, relapsed / refractory DLBCL, FL, lymphocyte-predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), and indolent NHL (including relapsed indolent NHL). This invention pertains to rituximab-refractory non-NHL leukemias, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloid leukemia; mantle cell lymphoma; and other hematologic malignancies. These malignancies can be treated with antibodies targeting B cell surface markers such as CD79b. This invention envisions treating such diseases by administering antibodies targeting B cell surface markers such as CD79b, including administering unconjugated (“naked”) antibodies or antibodies conjugated to the cytotoxic agents disclosed herein. This invention also envisions treating such diseases through combination therapies comprising concurrent or sequential administration of the anti-CD79b antibody or anti-CD79b antibody-drug conjugate described herein with another antibody or antibody-drug conjugate, another cytotoxic agent, radiation therapy, or other treatments. In exemplary treatment methods described herein, the anti-CD79b antibody described herein is administered together or sequentially with an anti-CD20 antibody, immunoglobulin, or a CD20-binding fragment thereof. The anti-CD20 antibody may be a naked antibody or an antibody-drug conjugate. In one embodiment of the combination therapy, the anti-CD79b antibody is the antibody described in this disclosure, and the anti-CD20 antibody is Rituxan®.

[0161] As used herein, the term “non-Hodgkin lymphoma” or “NHL” refers to cancers of the lymphatic system other than Hodgkin lymphoma. Typically, the distinction between Hodgkin lymphoma and non-Hodgkin lymphoma lies in the presence of Reed-Sternberg cells in Hodgkin lymphoma, which are absent in non-Hodgkin lymphoma. Examples of non-Hodgkin lymphoma as used herein include those identified by a person skilled in the art (e.g., an oncologist or pathologist) according to classification schemes known in the art, such as those identified according to the Revised European and American Lymphoma (REAL) classification scheme, as described in *Color Atlas of Clinical Hematology (3rd edition)* (Harcourt Publishers Ltd., 2000), edited by A. Victor Hoffbrand and John E. Pettit. See the lists in Figures 11.57, 11.58, and 11.59 for details. More specific examples include, but are not limited to: relapsed or refractory NHL, first-line low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-cell lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, lymph node marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasmacytic myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular lymphoma), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma. Large B-cell lymphoma, relapsed / refractory diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary lymphoma, high-grade immunoblastic NHL, high-grade lymphocytic NHL, high-grade non-cleavage small cell NHL, giant lesion NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis and / or Sezary syndrome, cutaneous (skin) lymphoma, anaplastic large cell lymphoma, angiogenic central lymphoma.

[0162] "Disease" means any disease that can be treated using the substances / molecules or methods described in this disclosure. It includes chronic and acute diseases or conditions, including those pathological conditions that predispose mammals to the disease. Non-limiting examples of treatable diseases described herein include: cancerous diseases such as malignant and benign tumors; non-leukemic and lymphoid malignancies; neurological, glial, astrocyte, hypothalamic and other glandular, macrophage, epithelial, stromal, and blastocystic diseases; and inflammatory, immune system, and other angiogenesis-related diseases. Diseases further include cancerous diseases such as B-cell proliferative disorders and / or B-cell tumors, such as: lymphoma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed-invasive NHL, relapsed-indolent NHL, refractory NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma.

[0163] The terms "cell proliferation disorder" and "proliferative disorder" refer to a condition associated with some degree of abnormal cell proliferation. In one embodiment, a proliferative disorder is cancer. In some embodiments, cancer is a B-cell proliferative disorder.

[0164] As used in this article, the term “tumor” refers to all proliferative cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.

[0165] "Treatment," "therapy," or "relief" refers to therapeutic treatments and preventative or protective measures aimed at preventing or slowing (alleviating) a target pathological disease or condition. Those requiring treatment include those already suffering from the condition, those susceptible to the condition, and those seeking to prevent it. If, according to the methods described in this disclosure, a subject or mammal expressing a CD79b peptide is successfully "treated" for cancer, the patient exhibits an observable and / or measurable reduction or absence of one or more of the following: a reduction in the number of cancer cells or the absence of cancer cells; a reduction in tumor size; inhibition (i.e., to some extent slowing and preferably stopping) of cancer cell infiltration into surrounding organs (including cancer spread to soft tissues and bones); inhibition (i.e., to some extent slowing and preferably stopping) of tumor metastasis; to some extent inhibiting tumor growth; and / or to some extent alleviating one or more symptoms associated with the specific cancer; a decrease in morbidity and mortality; and an improvement in quality of life. To some extent, the anti-CD79b antibody may prevent the growth of existing cancer cells or kill them; it may inhibit cell growth or be cytotoxic. The patient may also experience a reduction in these signs or symptoms.

[0166] The parameters mentioned above used to assess treatment success and disease improvement can be easily measured using routine procedures familiar to physicians. For cancer treatment, efficacy can be measured, for example, by assessing time to progression (TTP) and / or determining response rate (RR). Metastasis can be determined through staging tests, and bone scans along with calcium levels and other enzyme tests can determine if it has spread to the bones. CT scans can also be performed to check for spread to the pelvis and lymph nodes in that region. Lung and liver metastases are examined using chest X-rays and known liver enzyme level measurements, respectively. Other routine methods for monitoring disease include transrectal ultrasound (TRUS) and transrectal biopsy (TRNB).

[0167] For bladder cancer, which is a focal cancer, methods for determining disease progression include urinary cytology assessment via cystoscopy, monitoring for the presence of blood in the urine, examination of the urothelial lining via ultrasound or intravenous pyelography, computed tomography (CT), and magnetic resonance imaging (MRI). The presence of distant metastases can be assessed via abdominal CT, chest X-ray, or radionuclide imaging.

[0168] "Chronic" dosing refers to administration in a continuous manner, as opposed to the acute mode, in order to maintain the initial therapeutic effect (activity) for a longer period of time. "Intermittent" dosing is not a continuous, uninterrupted treatment, but rather a periodic treatment in nature.

[0169] "Individual," "subject," or "patient" refers to a vertebrate. In some embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock (such as cattle), locomotor animals, pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, the mammal is a human.

[0170] "Combined" administration with one or more other therapeutic agents includes simultaneous (concurrent) administration as well as administration in any order.

[0171] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers are typically pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0172] The terms "pharmaceutical composition" or "pharmaceutical formulation" refer to an antiCD79b immunoconjugate formulation in a biologically effective form of the permitted active ingredient and free of any additional components that would have unacceptable toxicity to the subject to which the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (carriers, additives) are excipients for which the active ingredient is reasonably administered to mammals to provide an effective dose.

[0173] "Sterile" preparations are sterile or contain no or substantially no live microorganisms and their spores.

[0174] The “effective amount” of the anti-CD79b immunoconjugate disclosed herein is an amount sufficient to achieve a specific purpose. The “effective amount” can be determined empirically and in a conventional manner relative to the stated purpose.

[0175] The term “therapeutic effective dose” refers to the amount of an anti-CD79b immunoconjugate that effectively “treats” a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective dose of the immunoconjugate may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow and preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., to some extent slow and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more cancer-related symptoms to some extent. See the definition of “therapeutic” in this document. To some extent, the immunoconjugate may prevent the growth of and / or kill existing cancer cells; it may inhibit cell growth and / or be cytotoxic. A “preventative effective dose” refers to the amount that effectively achieves the desired preventative outcome at the necessary dose and for the required period of time. A preventative effective dose is usually, but not necessarily, less than a therapeutically effective dose, as preventative doses are used on subjects before the onset of disease or in the early stages of disease.

[0176] The "growth inhibition level" of an anti-CD79b immunoconjugate is the amount of cell growth, particularly that of tumors such as cancer cells, that can be inhibited in vitro or in vivo. The "growth inhibition level" of an anti-CD79b immunoconjugate for inhibiting tumor cell growth can be determined empirically and through conventional methods.

[0177] The "cytotoxicity level" of an anti-CD79b immunoconjugate is the amount of cell damage it can cause, particularly tumor cells such as cancer cells, in vitro or in vivo. The "cytotoxicity level" of an anti-CD79b antibody for inhibiting tumor cell growth can be determined empirically and through conventional methods.

[0178] "Cells expressing CD79b" are cells that express endogenous or transfected CD79b peptides on their cell surface or in a secretory form. "Cancer expressing CD79b" is a cancer comprising cells that have CD79b peptides present on their cell surface or that produce and secrete CD79b peptides. "Cancer expressing CD79b" optionally produces sufficient levels of CD79b peptides on its cell surface such that anti-CD79b antibodies can bind to them and have a therapeutic effect on the cancer. In another embodiment, "cancer expressing CD79b" optionally produces and secretes sufficient levels of CD79b peptides such that anti-CD79b antibody antagonists bind to them and have a therapeutic effect on the cancer. Regarding the latter, the antagonist may be an antisense oligonucleotide that reduces, inhibits, or prevents the production and secretion of CD79b peptides secreted by tumor cells. Cancers "overexpressing" CD79b peptides are cancers that have higher levels of CD79b peptides on their cell surface or produce and secrete the peptide compared to non-cancer cells of the same tissue type. Such overexpression may be caused by gene amplification or increased transcription or translation. Overexpression of the CD79b peptide can be determined in detection or prognostic assays by assessing elevated levels of CD79b protein present on the cell surface or secreted by cells (e.g., by immunohistochemical assays using anti-CD79b antibodies against isolated CD79b peptides (which can be prepared from isolated nucleic acids encoding CD79b peptides using recombinant DNA technology); by FACS analysis, etc.). Alternatively or additionally, the level of nucleic acid or mRNA encoding the CD79b peptide in cells can be measured by fluorescence in situ hybridization (FISH, see WO98 / 45479, published October 1998; Southern hybridization, Northern hybridization, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR)). Antibody-based assays can also be used to study the overexpression of the CD79b peptide by measuring exfoliated antigens in biological fluids such as serum (see, for example: U.S. Patent No. 4,933,294, issued June 12, 1990; WO91 / 05264, published April 18, 1991; U.S. Patent No. 5,401,638, issued March 28, 1995; and Sias et al., *Journal of Immunology* 132:73-80 (1990)). In addition to the assays described above, a variety of in vivo assays can be performed by skilled practitioners.For example, cells in a patient's body can be exposed to antibodies, which may optionally be labeled with a detectable marker (such as a radioactive isotope), and the binding of the antibody to the patient's cells can be assessed, for example by external radiation scanning or by analyzing biopsy samples obtained from patients previously exposed to the antibody.

[0179] As used herein, the term "cytotoxic agent" refers to a pharmaceutical molecule that inhibits or prevents cell function and / or causes cell damage. The term is intended to include toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants capable of adversely affecting cell growth or proliferation. In one embodiment, the cytotoxic agent is monomethylolpropionate E (MMAE).

[0180] As used herein, a "lyophilized" composition refers to a liquid composition that has been freeze-dried to form a "mass". In some embodiments, the freeze-dried mass remains stable under storage conditions (5°C ± 3°C, protected from light) without significant changes in its structure, color, appearance, or moisture content. In one specific embodiment, the freeze-dried mass is smooth and free of dents. In some embodiments of this disclosure, the freeze-dried composition remains stable for about 60 months. In some embodiments of this disclosure, the freeze-dried composition remains stable for about 48 months.

[0181] A “stable” formulation is one in which the immunoconjugate substantially retains its physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity during storage. The storage period is typically selected based on the expected shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in, for example, the following literature: *Peptide and Protein Drug Delivery*, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991); and Jones, A., *Advanced Drug Delivery Review* 10: 29-90 (1993). Stability can be measured at a selected temperature over a selected time period. Stability can be assessed qualitatively and / or quantitatively in various ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary band electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis for comparing reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; and assessing antibody bioactivity or antigen-binding function. Instability may involve one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shearing / hydrolysis / fragmentation (e.g., hinge fragmentation), succinimide formation, unpaired cysteine ​​residues, N-terminal extension, C-terminal treatment, differential glycosylation, etc.

[0182] If, by visual inspection of color and / or transparency, or by ultraviolet scattering, or by size exclusion chromatography, the immunoconjugate shows no “indications” of aggregation, precipitation, and / or denaturation, or shows very little aggregation, precipitation, and / or denaturation, then the immunoconjugate is considered to “maintain its physical stability” in the pharmaceutical formulation.

[0183] If the chemical stability over a given time is such that the immunoconjugate is considered to retain its biological activity (as defined below), then the immunoconjugate is considered to “remain chemically stable” in a pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein moiety of the immunoconjugate (e.g., antibody). Chemical alterations may involve size changes (e.g., shearing), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., changes due to deamidation), which can be assessed using, for example, ion exchange chromatography or icIEF. Additionally or alternatively, the chemical stability of the immunoconjugate can be assessed by detecting and quantifying chemically altered forms of the pharmaceutical moiety of the immunoconjugate. Additionally or alternatively, the chemical stability of the immunoconjugate can be assessed by measuring the drug:antibody ratio (DAR), for example, by determining the DAR distribution in a composition containing the immunoconjugate using hydrophobic interaction liquid chromatography (HIC).

[0184] If the protein portion of an immunoconjugate (e.g., an antibody such as an anti-CD79b antibody) exhibits at least about 60% of the biological activity (within the assay error range) at a given time as observed when preparing a pharmaceutical formulation containing the immunoconjugate, then the antibody of the immunoconjugate is considered to "retain its biological activity" in the pharmaceutical formulation, as determined by an assay (e.g., an antigen binding assay).

[0185] The “deamidated” monoclonal antibodies described in this article are antibodies in which one or more asparagine residues are derived, for example, aspartic acid or isoaspartic acid.

[0186] The “oxidized” monoclonal antibody described in this article is an antibody in which one or more tryptophan residues and / or one or more methionine residues have been oxidized.

[0187] The "glycosylated" monoclonal antibody described in this article is an antibody in which one or more lysine residues have been glycosylated.

[0188] An antibody that is “easily deamidated” is an antibody that contains one or more residues that have been found to be easily deamidated.

[0189] An antibody that is “easily oxidized” is an antibody that contains one or more residues that have been found to be easily oxidized.

[0190] "Aggregative" antibodies are those that are found to aggregate with other antibody molecules, especially when aggregation occurs under freezing and / or stirring conditions.

[0191] "Easy to fragment" antibodies are, for example, antibodies that can be cut into two or more fragments in their hinge region.

[0192] Compared to monoclonal antibodies formulated in different formulations, “reducing deamidation, oxidation, aggregation or fragmentation” aims to prevent or reduce the amount of deamidation, oxidation, aggregation or fragmentation.

[0193] The formulated antibodies are preferably substantially pure and, preferably, substantially homogeneous (e.g., free from contaminating proteins). "Substantially pure" antibody means a composition containing at least about 90% by weight, preferably at least about 95% by weight, of antibody based on the total weight of proteins in the composition. "Substantially homogeneous" antibody means a composition containing at least about 99% by weight of antibody based on the total weight of proteins in the composition.

[0194] The term "isotonic" refers to a formulation of interest having an osmotic pressure substantially the same as that of human blood. The osmotic pressure of isotonic formulations is typically between approximately 250 and 350 mOsm. Isotonicity can be determined, for example, using a vapor pressure or a frozen osmometer.

[0195] As used herein, a "buffer" refers to a buffer solution that is resistant to pH changes through the action of its acid-base conjugate components. Non-limiting examples of buffers described herein include histidine, sodium phosphate, and sodium succinate. Buffers described in this disclosure preferably have a pH in the range of about 4.5 to about 7.0, preferably about 5.0 to about 6.0. In one embodiment, the buffer has a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In one exemplary embodiment, the buffer has a pH of 5.3. For example, an example of a buffer with pH controlled within this range is sodium succinate.

[0196] As used in this article, "surfactant" refers to a surface activator, preferably a nonionic surfactant. Examples of surfactants described herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl, myristyl, linoleyl, or stearoyl sulfonyl betaine; lauryl, myristyl, linoleyl, or stearoyl sarcosine; linoleyl, myristyl, or hexadecyl betaine; lauroylaminopropyl, cocamidopropyl, linoleamide propyl, myristylpropyl, palmitopropyl, or isostearamidopropyl betaine (e.g., lauroamide propyl); myristylaminopropyl, palmitopropyl, or isostearamidopropyl-dimethylamine; sodium methylcocoyl or disodium methyloleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). In one embodiment, the surfactant described herein is polysorbate 20. In another embodiment, the surfactant described herein is polysorbate 80.

[0197] As used herein, “sugar” refers to soluble carbohydrates. Non-limiting examples of sugars include glucose, fructose, sucrose, trehalose, arginine, glycerol, proline, dextran, and sugar alcohols such as glycerol, mannitol, and sorbitol.

[0198] II. Pharmaceutical Composition

[0199] In one aspect, this disclosure provides a pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant, wherein the concentration of the surfactant is at least 0.06% w / v (i.e., 0.6 mg / mL) and wherein the anti-CD79b immunoconjugate comprises the formula:

[0200]

[0201] in:

[0202] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (e.g., between about 2 and about 5). Between, for example, approximately 3.5).

[0203] In some embodiments, the compositions disclosed herein are lyophilized products. In some embodiments, the compositions disclosed herein are reconstituted compositions, i.e., compositions that have been reconstituted from lyophilized blocks. In some embodiments, 20 mL glass vials (such as sealed vials) are provided containing the pharmaceutical compositions described herein (e.g., lyophilized or reconstituted compositions).

[0204] A. Immunoconjugates

[0205] This disclosure relates to immunoconjugates (interchangeably referred to as “antibody-drug conjugates” or ADCs) comprising antibodies conjugated to small molecule toxins, such as monomethyl ozoglustatin (MMAE) (a synthetic analog of ozoglustatin). This disclosure provides an anti-CD79b immunoconjugate having the formula Ab – (L – D)p, wherein Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); L The linker comprises 6-maleiminohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB); D is MMAE; and p-value is from about 1 to about 8 (e.g., between about 2 and about 5, for example, about 3.5); and wherein the anti-CD79b immunoconjugate has the following structure:

[0206] .

[0207] The anti-CD79b immunoconjugates described in this disclosure may refer to anti-CD79b-MC-val-cit-MMAE, anti-CD79b-MC-vc-MMAE, or anti-CD79b-vc-MMAE. An example of an anti-CD79b immunoconjugate described in this disclosure is polatuzumab vedotin. Polatuzumab vedotin has CAS Registry No. 1313206-42-6, IUPHAR / BPS No. 8404, and KEGG No. D10761. The terms “polatuzumab vedotin,” “DCDS4501A,” and “RG7596” encompass all corresponding immunoconjugates required to obtain marketing authorization for the same product or biosimilar product in countries or territories selected from the group consisting of the United States, Europe, and Japan.

[0208] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 30 mg / mL, or about 10 mg / mL to about 20 mg / mL. In one specific embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 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, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, or about 60 mg / mL.

[0209] In some embodiments of any of the embodiments provided herein, the “approximate” value or parameter covers an error range (including any range between these values) of any of the values ​​or parameters being ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0210] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is from about 10 mg / mL to about 20 mg / mL. In one exemplary embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is about 10 mg / mL. In another exemplary embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is about 20 mg / mL.

[0211] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 5 mg / mL to 60 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL, or 10 mg / mL to 20 mg / mL. In one specific embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 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, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, or 60 mg / mL.

[0212] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is from 10 mg / mL to 20 mg / mL. In one exemplary embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 10 mg / mL. In another exemplary embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 20 mg / mL.

[0213] As envisioned in this paper, the anti-CD79b immunoconjugate has the following formula I, wherein the anti-CD79b antibody or a fragment thereof (Ab) is conjugated (i.e. covalently linked) to the MMAE drug moiety (D) via a linker (L).

[0214]

[0215] In Formula I, p is the average number of drug portions per antibody, which can range from about 1 to about 20 drug portions per antibody, and in some embodiments, from 1 to about 8 drug portions per antibody. This disclosure includes a composition comprising a mixture of antibody-drug compounds of Formula I, wherein the average drug loading per antibody is from about 2 to about 5 or from about 3 to about 4, for example, 3.5.

[0216] 1. Anti-CD79b antibody

[0217] This disclosure provides immunoconjugates comprising an anti-CD79b antibody or a functional fragment thereof.

[0218] In one aspect, this disclosure provides an anti-CD79b antibody, which preferably specifically binds to CD79b. Optionally, the antibody is a monoclonal antibody, an antibody fragment (including Fab, Fab', F(ab')2, and Fv fragments), a bisomal antibody, a single-domain antibody, a chimeric antibody, a humanized antibody, a single-chain antibody, or an antibody that competitively inhibits the binding of an anti-CD79b polypeptide antibody to its corresponding antigenic epitope. The antibodies described in this disclosure may optionally be produced in CHO cells or bacterial cells, and preferably induce cell death in the cells to which they are bound.

[0219] This disclosure provides an anti-CD79b immunoconjugate comprising a humanized anti-CD79b antibody, wherein the monovalent affinity of the antibody for CD79b (e.g., the affinity of an antibody as a Fab fragment for CD79b) is substantially the same as the monovalent affinity of a mouse antibody (e.g., the affinity of a mouse antibody as a Fab fragment for CD79b) or the monovalent affinity of a chimeric antibody (e.g., the affinity of a chimeric antibody as a Fab fragment for CD79b).

[0220] In one embodiment, this disclosure provides an anti-CD79b immunoconjugate comprising a humanized anti-CD79b antibody, wherein the bivalent form of the antibody has an affinity for CD79b (e.g., the affinity of an antibody as IgG to CD79b) of 0.3 nM or better. In another embodiment, this disclosure provides a humanized anti-CD79b antibody, wherein the bivalent form of the antibody has an affinity for CD79b (e.g., the affinity of an antibody as IgG to CD79b) of 0.5 nM. In yet another embodiment, this disclosure provides a humanized anti-CD79b antibody, wherein the bivalent form of the antibody has an affinity for CD79b (e.g., the affinity of an antibody as IgG to CD79b) of 0.5 + / - 0.1 nM. In another embodiment, this disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the bivalent antibody for CD79b (e.g., the affinity of an antibody as IgG for CD79b) is between 0.3 nM and 0.7 nM. In another embodiment, this disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the bivalent antibody for CD79b (e.g., the affinity of an antibody as IgG for CD79b) is between 0.4 nM and 0.6 nM. In another embodiment, this disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the bivalent antibody for CD79b (e.g., the affinity of an antibody as IgG for CD79b) is between 0.5 nM and 0.55 nM.

[0221] As is recognized in the art, the binding affinity of a ligand to its receptor can be determined using any of a variety of assays and expressed in various quantitative values. Therefore, in one embodiment, binding affinity is expressed as a Kd value and reflects intrinsic binding affinity (e.g., minimized affinity effects). Typically and preferably, binding affinity is measured in vitro, whether in a cell-free or cell-associated environment. As detailed herein, fold differences in binding affinity can be quantified by the ratio of the monovalent binding affinity value of a humanized antibody (e.g., in Fab form) to that of a reference / control antibody (e.g., in Fab form) (e.g., a mouse antibody with a donor hypervariable sequence), wherein the binding affinity values ​​are measured under similar assay conditions. Therefore, in one embodiment, the fold difference in binding affinity is determined as the ratio of the Kd value of the humanized antibody in Fab form to that of the reference / control Fab antibody. For example, in one embodiment, if the affinity of the antibody (A) described in this disclosure is "3 times lower" than that of the reference antibody (M), then when the Kd value of A is 3x, the Kd value of M is 1x, and the ratio of the Kd of A to the Kd of M is 3:1. Conversely, in one embodiment, if the affinity of the antibody (C) described in this disclosure is "3 times higher" than that of the reference antibody (R), then when the Kd value of C is 1x, the Kd value of R is 3x, and the ratio of the Kd of C to the Kd of R is 1:3. Any of a variety of assays known in the art, including those described herein, can be used to obtain binding affinity assay results; these methods include, for example, Biacore, radioimmunoassay (RIA), and ELISA.

[0222] The provided anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6).

[0223] In some embodiments of this disclosure, the anti-CD79b antibody comprises a heavy chain variable domain (VH) containing the following amino acid sequence:

[0224] EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSS (SEQ ID NO: 7)

[0225] And a light chain variable domain (VL) containing the following amino acid sequence:

[0226] DIQLTQSPSSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIKR (SEQ ID NO: 8).

[0227] In some embodiments, the heavy chain comprises the following amino acid sequence:

[0228] EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 9)

[0229] And a light chain containing the following amino acid sequence:

[0230] DIQLTQSPSSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 10).

[0231] 2. Connector

[0232] This disclosure provides a connector comprising a variety of components, including 6-maleimide hexanoyl (“MC”), valine-citrulline (“val-cit” or “vc”), and p-aminobenzyloxycarbonyl (“PAB”). Such connector components are known in the art and are briefly described below.

[0233] As used herein, the connector has Formula II:

[0234]

[0235] Where A is the stretching unit, a is an integer from 0 to 1; W is the amino acid unit, w is an integer from 0 to 12; Y is the spacer unit, and y is 0, 1, or 2. The "stretching unit" links the antibody to another linker component. The stretching unit MC provided in this paper is shown below (where the wavy line indicates the site covalently linked to the antibody):

[0236] MC

[0237] Amino acid units enable proteases to cleave linkers, thereby facilitating the release of drugs from immunoconjugates upon exposure to intracellular proteases such as lysosomal enzymes. See, for example, Doronina et al. (2003), *Nature Biotechnol.* 21: 778-784. Amino acid units may contain naturally occurring amino acid residues as well as minor amino acids and non-natural amino acid analogs such as citrulline. The selectivity of the amino acid unit can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminogen activator. The amino acid unit described herein is a valine-citrulline (VC or val-cit) dipeptide.

[0238] As used herein, the "spacer" unit is a suicide unit, and the antibody is linked to the drug moiety via stretching and amino acid units. The "suicide" spacer unit releases the drug moiety without a separate hydrolysis step due to the amide bond and the formation of a carbamate, methyl carbamate, or carbonate between benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al. (2005), Expert Opinion. Ther. Patents (2005) 15: 1087-1103. The spacer unit provided herein is p-aminobenzyloxycarbonyl (PAB).

[0239] The linker components, including stretching units, spacer units, and amino acid units, can be synthesized by methods known in the art, such as those described in US 2005-0238649A1.

[0240] The connector described in this disclosure is MC-val-cit-PAB, as shown below.

[0241]

[0242] 3. Monomethylolpropionate (MMAE)

[0243] The immunoconjugates described in this disclosure include anti-CD79b antibodies or fragments thereof conjugated to olistatin (a synthetic analogue of scabra), a biosynthetic analogue of olistatin (US Patent Nos. 5635483; 5780588). Previous studies have shown that scabra and olistatin interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., (2001), *Antimicrob. Agents and Chemother.* 45(12): 3580-3584), and possess anticancer activity (US Patent No. 5663149) and antifungal activity (Pettit et al., (1998), *Antimicrob. Agents and Chemother.* 42: 2961-2965). The olistatin drug moiety can be linked to the antibody via the N-terminus or C-terminus of the peptide drug moiety (WO 02 / 088172). The orestatin described in this disclosure is a synthetic, uncharged, N-terminally linked monomethyl orestatin (MMAE) pentapeptide, as shown below.

[0244]

[0245] The anti-CD79b immunoconjugate shown in Formula I comprises an anti-CD79b antibody or a fragment thereof, a linker having various linker components, and MMAE, as abbreviated below, wherein “Ab” is its anti-CD79b antibody or a fragment thereof; p is 1 to about 8 (e.g., between about 2 and about 5, such as about 3.5); “Val-Cit” or “vc” is a valine-citrulline dipeptide; and “S” is a sulfur atom.

[0246]

[0247] It should be noted that in some structural descriptions of sulfur-linked immunoconjugates herein, the designation "Ab-S" antibody is used only to indicate the sulfur-linking feature and is not intended to indicate that a specific sulfur atom carries multiple linker-drug moieties. The left parenthesis in the following structures can also be placed to the left of the sulfur atom between Ab and S, which would be an equivalent description of the immunoconjugates disclosed herein.

[0248] 4. Drug loading

[0249] Drug loading is denoted by p, which is the average number of drug moieties in each antibody in the molecule shown in Formula I. The drug loading of each antibody ranges from 1 to 20 drug moieties (D). The ADC shown in Formula I comprises an antibody set conjugated to drug moieties ranging from 1 to 20. The average number of drug moieties in each antibody in the ADC formulation obtained by the conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADC, denoted by p, can also be determined. In some cases, where p is a specific value obtained from ADCs with other drug loadings, the separation, purification, and characterization of homogeneous ADCs can be achieved by methods such as reversed-phase HPLC or electrophoresis. Therefore, the drug formulation of the antibody-drug conjugate shown in Formula I can be a heterogeneous mixture of such conjugates and antibodies linked to 1, 2, 3, 4, or more drug moieties.

[0250] For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, in the embodiments provided herein, when the linker is a cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups, or only one or a few thiol groups with sufficiently high reactivity to connect the linker. In some embodiments, higher drug loading, such as p > 5, may lead to aggregation, insolubility, toxicity, or decreased cell permeability of some antibody-drug conjugates. In some embodiments, the drug loading of the ADCs described in this disclosure ranges from 1 to about 8, from about 2 to about 6, from about 3 to about 5, or from about 3.5. Indeed, studies have shown that for some ADCs, the optimal proportion of the drug portion per antibody may be less than 8, and may be from about 2 to about 5. See US 2005-0238649 A1.

[0251] In some embodiments, the amount of drug moiety bound to the antibody during the coupling reaction is less than the theoretical maximum. The antibody may contain, for example, lysine residues that do not react with drug-linker intermediates or linker reagents, as described below. Typically, the antibody does not contain many free and reactive cysteine ​​thiols that could be linked to the drug moiety; in fact, most cysteine ​​thiols in the antibody are present in disulfide bond form. In some embodiments, the antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partially or completely reducing conditions to generate reactive cysteine ​​thiols. In some embodiments, the antibody is denatured to reveal reactive nucleophilic groups such as lysine or cysteine.

[0252] The drug loading (drug / antibody ratio) of an ADC can be controlled in various ways, such as: (i) limiting the molar excess of the drug-adaptor intermediate or adaptor reagent relative to the antibody, (ii) limiting the coupling reaction time or temperature, and (iii) partially or limiting the reduction conditions of cysteine ​​thiol modification.

[0253] It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or sequentially with a linker reagent and a drug moiety reagent, the resulting product is a mixture of ADC compounds having a distribution of one or more drug moieties linked to an antibody. The average amount of drug in each antibody can be calculated from the mixture using a dual ELISA antibody assay, which is specific for both the antibody and the drug. Individual ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, for example: McDonald et al. (2006), *Protein Engineering, Design & Selection* 19(7): 299-307; Hamblett et al. (2004), *Clin. Cancer Res.* 10:7063-7070; Hamblett, KJ et al., “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate”, Abstract No. 624, American Association for Cancer Research 2004 Annual Meeting, March 27-31, 2004, AACR Proceedings, Vol. 45, March 2004; Alley, SC Others, “Controlling the Position of Drug Attachment in Antibody-Drug Conjugates,” Abstract No. 627, American Association for Cancer Research 2004 Annual Meeting, March 27-31, 2004, AACR Proceedings, Vol. 45, March 2004. In some embodiments, homogeneous ADCs with a single drug loading can be separated from the conjugation mixture by electrophoresis or chromatography.

[0254] 5. Exemplary methods for preparing immunoconjugates

[0255] The ADC shown in Formula I can be prepared via a variety of pathways using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting the nucleophilic group of the antibody with a divalent linker reagent to form Ab-L via a covalent bond, and then reacting it with the drug moiety D; and (2) reacting the nucleophilic group of the drug moiety with a divalent linker reagent to form DL via a covalent bond, and then reacting it with the nucleophilic group of the antibody. An exemplary method for preparing the ADC shown in Formula I via the latter pathway is described in US 2005-0238649 A1, which is expressly incorporated herein by reference.

[0256] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amino groups, (ii) side-chain amino groups, such as lysine, (iii) side-chain thiols, such as cysteine, and (iv) glycosylated hydroxyl or amino groups where the antibody undergoes glycosylation. The amino, thiol, and hydroxyl groups are nucleophilic groups capable of reacting with electrophilic groups on the linker moiety and linker reagent to form covalent bonds. These electrophilic groups include: (i) reactive esters, such as NHS esters, HOBt esters, halocarboxylates, and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleamide groups. Some antibodies have reducible interchain disulfide bonds, i.e., cysteine ​​bridges. Treatment with reducing agents such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) allows the antibody to react with the linker reagent, resulting in complete or partial reduction of the antibody. Therefore, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by modifying lysine residues, for example, by reacting lysine residues with 2-iminothiacyclopentane (Traut reagent) to convert amines into thiols. Reactive thiol groups can be introduced into antibodies by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing mutant antibodies containing one or more non-natural cysteine ​​amino acid residues).

[0257] The antibody-drug conjugates described in this disclosure can also be prepared by reacting an electrophilic group on the antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug. Nucleophilic groups available on the linker reagent include, but are not limited to, acylhydrazides, oximes, amino groups, hydrazides, thioaminoureas, hydrazide carboxylates, and aryl acylhydrazides. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on the linker reagent or drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example with a periodate oxidizing agent, to form an aldehyde or ketone group, which can react with an amino group on the linker reagent or drug moiety. The resulting imine Schiff base group can form a stable link or can be reduced, for example by a borohydride reagent, to form a stable amine bond. In one embodiment, the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium periodate can generate carbonyl (aldehyde and ketone) groups in the antibody, which can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can react with sodium periodate to produce an aldehyde in place of the first amino acid (Geoghegan and Stroh (1992), Bioconjugate Chem. 3: 138-146; US 5362852). Such aldehydes can react with drug moieties or linker nucleophiles.

[0258] The nucleophilic groups on the pharmaceutical moiety include, but are not limited to, amine, mercapto, hydroxyl, acylhydrazine, oxime, hydrazine, thioaminourea, hydrazide ester, and aryl acylhydrazine groups, which are capable of reacting with electrophilic groups on the linker moiety and the linker reagent to form covalent bonds. The linker reagent includes, (i) active esters, such as NHS esters, HOBt esters, halocarboxyl esters, and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleamide groups.

[0259] The compounds described in this disclosure are explicitly envisioned, but not limited to, ADCs prepared using the following crosslinking agents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, and sulfon-SMPB, as well as commercially available SVSB (succinimide (4-vinyl sulfone) benzoate) (e.g., available from Pierce Biotechnology, Inc. (Rockford, IL., USA); see 2003-2004 Applications Handbook and Catalog, pp. 467-498).

[0260] Various bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imino esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and di-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene), can also be used to prepare immunoconjugates containing antibodies and cytotoxic agents. For example, ricin immunotoxin can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO94 / 11026.

[0261] B. Surfactants

[0262] This disclosure provides a composition comprising an anti-CD79b immunoconjugate and a surfactant. In some embodiments, the surfactant is polysorbate (e.g., polysorbate 20 and polysorbate 80); poloxamer (e.g., poloxamer 188); N-octyl-β-D-glucopyranoside (OG); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl, myristyl, linoleyl, or stearoyl sulfobetaine; lauryl, myristyl, linoleyl, or stearoyl sarcosine; linoleyl, myristyl, or hexadecyl betaine; lauroylaminopropyl, cocamidopropyl, linoleamide propyl, myristylpropyl, palmitopropyl, or isostearamidopropyl betaine (e.g., lauroamide propyl); myristylaminopropyl, palmitopropyl, or isostearamidopropyl-dimethylamine; sodium methylcocoyl or disodium methyl oleyl-taurate; and MONAQUAT™ Series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol such as Pluronics or PF68; or any combination thereof.

[0263] In some embodiments, the surfactant described herein is a nonionic surfactant. In some exemplary embodiments, the surfactant is selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188 (P188), N-octyl-β-D-glucopyranoside (OG), and combinations thereof. In one specific embodiment, the surfactant is PS20. In another embodiment, the surfactant described herein is PS80.

[0264] In some embodiments, the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least about 0.01% w / v (i.e., 0.1 mg / mL) and about 0.20% w / v (i.e., 2.0 mg / mL). In some embodiments, the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least about 0.01% w / v (i.e., 0.1 mg / mL), at least about 0.02% w / v (i.e., 0.2 mg / mL), at least about 0.03% w / v (i.e., 0.3 mg / mL), at least about 0.04% w / v (i.e., 0.4 mg / mL), at least about 0.05% w / v (i.e., 0.5 mg / mL), at least about 0.06% w / v (i.e., 0.6 mg / mL), at least about 0.07% w / v (i.e., 0.7 mg / mL), at least about 0.08% w / v (i.e., 0.8 mg / mL), at least about 0.09% w / v (i.e., 0.9 mg / mL), at least about 0.10% w / v (i.e., 1 mg / mL), at least about 0.11% w / v (i.e., 1.1 mg / mL), and at least about 0.12% w / v (i.e., 1.2 mg / mL). The concentrations are at least about 0.13% w / v (i.e., 1.3 mg / mL), at least about 0.14% w / v (i.e., 1.4 mg / mL), at least about 0.15% w / v (i.e., 1.5 mg / mL), at least about 0.16% w / v (i.e., 1.6 mg / mL), at least about 0.17% w / v (i.e., 1.7 mg / mL), at least about 0.18% w / v (i.e., 1.8 mg / mL), at least about 0.19% w / v (i.e., 1.9 mg / mL), or at least about 0.20% w / v (i.e., 2.0 mg / mL), including any range between these values. In one specific embodiment, the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least about 0.06% w / v (i.e., 0.6 mg / mL). In another specific embodiment, the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least about 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C-8°C for at least about 24, 48, or 72 hours.

[0265] In some embodiments, the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least about 0.01% w / v (i.e., 0.1 mg / mL) and about 0.20% w / v (i.e., 2.0 mg / mL). In some embodiments, the concentration of the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at least 0.01% w / v, at least 0.02% w / v, at least 0.03% w / v, at least 0.04% w / v, at least 0.05% w / v, at least 0.06% w / v, at least 0.07% w / v, at least 0.08% w / v, at least 0.09% w / v, at least 0.10% w / v, at least 0.11% w / v, at least 0.12% w / v, at least 0.13% w / v, at least 0.14% w / v, at least 0.15% w / v, at least 0.16% w / v, at least 0.17% w / v, at least 0.18% w / v, at least 0.19% w / v, or at least 0.20% w / v, including any range between these values. In one embodiment, the concentration of the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at least 0.06% w / v. In another embodiment, the concentration of the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at least 0.12% w / v. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized block. In some embodiments, the lyophilized pharmaceutical composition is contained in a glass vial.

[0266] In some embodiments, the concentration of a surfactant (e.g., PS20) in the liquid pharmaceutical composition is at least about 0.1 mg / mL to about 2.0 mg / mL. In some embodiments, the concentration of a surfactant (e.g., PS20) in the liquid pharmaceutical composition is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / mL (inclusive of any range between these values). In one specific embodiment, the concentration of a surfactant (e.g., PS20) in the liquid pharmaceutical composition is at least about 0.6% mg / mL. In another specific embodiment, the concentration of a surfactant (e.g., PS20) in the liquid pharmaceutical composition is at least 1.2 mg / mL. In some embodiments, the liquid pharmaceutical composition is reconstituted from a lyophilized pharmaceutical composition (such as a lyophilized block) (e.g., using SWFI reconstitution). In some embodiments, the liquid pharmaceutical composition is contained in a glass vial. In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C-8°C for at least about 24, 48, or 72 hours.

[0267] In one exemplary embodiment, the concentration of the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is 0.06% w / v. In another exemplary embodiment, the concentration of the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is 0.12% w / v. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized block. In some embodiments, the lyophilized pharmaceutical composition is contained in a glass vial.

[0268] In one exemplary embodiment, the concentration of a surfactant (e.g., PS20) in the liquid pharmaceutical composition is 0.6 mg / mL. In another exemplary embodiment, the concentration of a surfactant (e.g., PS20) in the liquid pharmaceutical composition is 1.2 mg / mL. In some embodiments, the lyophilized composition (such as lyophilized blocks) of the liquid pharmaceutical composition is reconstituted (e.g., using SWFI). In some embodiments, the liquid pharmaceutical composition is contained in a glass vial. In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C–8°C for at least about 24, 48, or 72 hours. In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C-8°C for at least about 24, 48, or 72 hours.

[0269] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 10 mg / mL, and the concentration of the surfactant in the pharmaceutical composition is at least 0.06% w / v (i.e., 0.6 mg / mL). In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 10 mg / mL, and the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the pharmaceutical composition is a reconstituted composition (reconstituted from a lyophilized composition using SWFI). In some embodiments, the pharmaceutical composition is contained in a glass vial. In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C–8°C for at least about 24, 48, or 72 hours.

[0270] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 10 mg / mL, and the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is 0.06% w / v (i.e., 0.6 mg / mL). In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 10 mg / mL, and the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the pharmaceutical composition is a reconstituted composition (reconstituted from a lyophilized composition using SWFI). In some embodiments, the pharmaceutical composition is contained in a glass vial.

[0271] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 20 mg / mL, and the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least 0.06% w / v (i.e., 0.6 mg / mL). In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 20 mg / mL, and the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is at least 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the pharmaceutical composition is a reconstituted composition (reconstituted from a lyophilized composition using SWFI). In some embodiments, the pharmaceutical composition is contained in a glass vial.

[0272] In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 20 mg / mL, and the concentration of the surfactant (e.g., PS20) in the pharmaceutical composition is 0.06% w / v (i.e., 0.6 mg / mL). In some embodiments, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 20 mg / mL, and the concentration of the surfactant in the pharmaceutical composition is 0.12% w / v (i.e., 1.2 mg / mL). In some embodiments, the liquid pharmaceutical composition is stably stored at 30°C for at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or stably stored at 2°C–8°C for at least about 24, 48, or 72 hours.

[0273] In some embodiments, the pharmaceutical composition is a liquid composition suitable for intravenous administration. In some embodiments, the liquid composition suitable for administration comprises a surfactant (e.g., polysorbate 20) at a concentration of about 0.0432 to about 0.162 mg / mL. In some embodiments, the liquid composition comprises polatuzumab vedotin at a concentration between about 0.72 mg / mL and about 2.7 mg / mL. In some embodiments, the liquid composition suitable for intravenous administration comprises about 0.72 mg / mL polatuzumab vedotin and about 0.0432 mg / mL polysorbate 20. In some embodiments, the liquid composition suitable for intravenous administration comprises about 2.7 mg / mL polatuzumab vedotin and about 0.162 mg / mL polysorbate 20.

[0274] C. Buffer

[0275] According to this disclosure, pharmaceutical compositions comprising buffers are provided. Not wishing to be bound by theory, the buffers are used to maintain the pH of the pharmaceutical composition during its production, storage, and use. Non-limiting examples of buffers include histamine, succinate, sodium succinate, sodium bicarbonate, calcium chloride, magnesium sulfate, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (Bicine), 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (Tricine), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 2- [4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazindiethanesulfonic acid (PIPES), dimethylarsonic acid, 2-morpholino-4-ylethanesulfonic acid (MES), or phosphate-buffered saline (PBS). Other suitable buffers may be acetate, citrate, borate, and phosphate.

[0276] In exemplary embodiments of this disclosure, the buffer is a histidine buffer or a succinate buffer. In one specific embodiment, the succinate buffer is a sodium succinate buffer.

[0277] In some embodiments, the concentration of the buffer is from about 10 mM to about 200 mM. In one specific embodiment, the concentration of the buffer is approximately 10 mM, approximately 11 mM, approximately 12 mM, approximately 13 mM, approximately 14 mM, approximately 15 mM, approximately 16 mM, approximately 17 mM, approximately 18 mM, approximately 19 mM, approximately 20 mM, approximately 21 mM, approximately 22 mM, approximately 23 mM, approximately 24 mM, approximately 25 mM, approximately 26 mM, approximately 27 mM, approximately 28 mM, approximately 29 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, approximately 90 mM, approximately 95 mM, approximately 100 mM, approximately 105 mM, approximately 110 mM, approximately 115 mM, approximately 120 mM. mM, approximately 125 mM, approximately 130 mM, approximately 135 mM, approximately 140 mM, approximately 145 mM, approximately 150 mM, approximately 155 mM, approximately 160 mM, approximately 165 mM, approximately 170 mM, approximately 175 mM, approximately 180 mM, approximately 185 mM, approximately 190 mM, approximately 195 mM, or approximately 200 mM.

[0278] In another specific embodiment, the concentration of the buffer is 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 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. mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, about 190mM, 195mM or 200mM.

[0279] In one exemplary embodiment of this disclosure, the concentration of the sodium succinate buffer is from about 10 mM to about 200 mM. In one embodiment, the concentration of the sodium succinate buffer is about 10 mM. In another embodiment, the concentration of the sodium succinate buffer is 10 mM.

[0280] In some embodiments, the compositions of this disclosure have a pH of about 5.0 to about 6.0. In specific embodiments, the buffer solution has a pH of 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, or about 6.0. In other specific embodiments, the buffer solution has a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0281] In one exemplary embodiment, the composition of this disclosure has a pH of about 5.3. In another exemplary embodiment, the composition of this disclosure has a pH of 5.3.

[0282] D. Sugar

[0283] According to this disclosure, pharmaceutical compositions comprising sugars are provided. Without being bound by theory, sugars are used to facilitate cryoprotection of the pharmaceutical composition, thereby preventing aggregation and maintaining its chemical and physical stability. In some embodiments, the sugar is glucose, fructose, sucrose, trehalose, arginine, glycerol, proline, dextran, dextran 40, glycerol, mannitol, or sorbitol.

[0284] In some embodiments, the sugar is selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, dextran 40, and trehalose. In one exemplary embodiment, the sugar is sucrose.

[0285] In some embodiments, the sugar concentration is from about 50 mM to about 300 mM. In one specific embodiment, the buffer concentration is about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about 300 mM. In some embodiments, the sugar concentration is from about 100 mM to about 260 mM.

[0286] In some embodiments, the sugar concentration is from 50 mM to 300 mM. In one specific embodiment, the buffer concentration is 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some embodiments, the sugar concentration is from 100 mM to 260 mM.

[0287] In one exemplary embodiment of this disclosure, the concentration of sucrose is about 120 mM. In another exemplary embodiment, the concentration of sucrose is 120 mM.

[0288] In another aspect, this disclosure provides a pharmaceutical composition prepared by lyophilization of a liquid composition comprising 20 mg / mL of an anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v of polysorbate 20, and 120 mM sucrose, wherein the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate has the formula:

[0289]

[0290] in:

[0291] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (e.g., between about 2 and about 5). Between, for example, approximately 3.5).

[0292] In some embodiments of this disclosure, the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0293] E. Stability

[0294] In some embodiments, the physical stability, chemical stability, or biological activity of the pharmaceutical composition in liquid or lyophilized form was evaluated or measured. Any method known in the art and described in the embodiments herein can be used to evaluate the stability and biological activity of the pharmaceutical compositions described in this disclosure. For example, the stability of antibodies in a pharmaceutical composition can be measured by methods including, but not limited to, size exclusion chromatography (SEC or SE-HPLC), imaging plasma isoelectric focusing (icIEF), peptide mapping analysis, high-intensity optical impedance spectroscopy (HIAC) determination, and capillary electrophoresis (CE) techniques such as CE-sodium dodecyl sulfate (CE-SDS) and CE-polysaccharide analysis. In some embodiments, a stable pharmaceutical composition (or formulation) is one in which the immunoconjugates substantially maintain their physical and / or chemical stability and / or biological activity after storage under specified conditions (such as temperature, relative humidity, residual moisture, presence or absence of light, and agitation for a period of time) (e.g., storage for a specified period of time or duration such as hours, days, months, years, etc.). In some embodiments, an immunoconjugate (and / or the antibody portion of an immunoconjugate, such as an anti-CD79b antibody) present in a pharmaceutical composition is considered to be biologically stable (e.g., retains its biological activity) if its biological activity is within the range acceptable to a national or regional drug regulatory agency (e.g., the U.S. Food and Drug Administration (FDA), the Australian Therapeutic Goods Administration (TGA), the European Medicines Agency (EMA), etc.) for a given period of time. For example, the biological activity of an immunoconjugate (such as an immunoconjugate containing an anti-CD79b antibody) or a pharmaceutical composition containing such an immunoconjugate can be measured by its ability to bind antigens (e.g., the ability of an anti-CD79b antibody to bind CD79b, such as human CD79b). Many assays known in the art can be used to measure antibody binding affinity parameters, including but not limited to, surface plasmon resonance (SPR), radioimmunoassay (RIA), and ELISA. Additionally or alternatively, the biological activity of an immunoconjugate (such as an immunoconjugate containing an anti-CD79b antibody) is measured by its ability (e.g., in vitro or in vivo) to inhibit cell growth and / or cell proliferation or (e.g., in vitro or in vivo) to induce cell death (including programmed cell death (apoptosis)). Further details regarding the determination of the biological activity of an anti-CD79b antibody or an immunoconjugate containing an anti-CD79b antibody are provided in U.S. Patent No. 8,088,387, the contents of which are incorporated herein by reference in their entirety.

[0295] In some embodiments, the pharmaceutical compositions disclosed herein remain stable at -20°C for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 3 years, at least about 4 years, or at least about 5 years.

[0296] In some embodiments, the pharmaceutical compositions disclosed herein remain stable for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 3 years, at least about 4 years, or at least about 5 years when stored protected from light at 2°C to 8°C (e.g., 5°C ± 3°C).

[0297] In some embodiments, the pharmaceutical compositions disclosed herein remain stable at 25°C to 40°C for at least about 30 minutes, at least about 60 minutes, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 1 day, at least about 2 days, or at least about 3 days.

[0298] In one exemplary embodiment, the composition remains stable for at least about 48 months when stored in the dark at 5°C ± 3°C. In another exemplary embodiment, the composition remains stable for about 48 months when stored in the dark at 5°C ± 3°C.

[0299] In some embodiments, the stability of the lyophilized pharmaceutical compositions (e.g., lyophilized blocks) described in this disclosure can be examined. In some embodiments, the lyophilized blocks show no significant changes in structure, color, appearance, or moisture content under storage conditions (5°C ± 3°C, protected from light). In one specific embodiment, the lyophilized blocks have a moisture content of less than 5% under storage conditions. In another specific embodiment, the lyophilized blocks are smooth and free of dents under storage conditions. In some embodiments of this disclosure, the lyophilized blocks remain stable under storage conditions for at least about 48 months.

[0300] In some embodiments, the stability of the pharmaceutical composition of this disclosure is determined by size exclusion high-performance liquid chromatography (SE-HPLC). In one exemplary embodiment, the composition has a main peak (peak area %) of at least 95.0 as determined by SE-HPLC.

[0301] In some embodiments, the lyophilized pharmaceutical compositions (such as blocks) provided herein are stably stored at temperatures between about 2°C and about 8°C for at least 44 months (such as at least about 30 months, 35 months, 40 months, 45 months, 50 months, or 55 months) with a residual moisture content ranging from about 0.3% (w / w) to about 3.2% (w / w), including any range between these values. In some embodiments, the lyophilized pharmaceutical compositions (such as blocks) provided herein are stably stored at about 25°C for at least 7 months (such as at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 8 months, or 10 months) with a residual moisture content ranging from about 0.3% (w / w) to about 3.2% (w / w), including any range between these values.

[0302] In some embodiments, the lyophilized pharmaceutical compositions (such as blocks) provided herein remain stable under the following conditions: (a) protein concentration between about 17 and about 23 mg / mL, (b) succinate concentration between about 7 and about 23 mM, (c) sucrose concentration between about 90 and about 150 mM, (d) polysorbate 20 concentration between about 0.9 and about 1.5 mg / mL, and (e) pH between about 4.95 and about 5.65. In some embodiments, a lyophilized pharmaceutical composition (such as a block) comprising one or more of (a) to (e) can remain stable at a temperature between about 2°C and about 8°C for up to a period of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 months.

[0303] G. Compositions for intravenous administration.

[0304] This disclosure provides pharmaceutical compositions and liquid compositions suitable for intravenous (IV) administration. The compositions intended for in vivo IV administration should be sterile. This can be easily achieved by filtering through a sterile filter membrane before or after the preparation of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions of this disclosure are reconstituted with sterile water for injection (SWFI). In some embodiments, the reconstituted composition is further diluted in an isotonic buffer solution in an intravenous (IV) infusion bag.

[0305] In one aspect, this disclosure provides a pharmaceutical composition comprising an anti-CD79b immunoconjugate, a surfactant, a succinate buffer, and a sugar, wherein the pharmaceutical composition, when reconstituted in water, forms a liquid pharmaceutical composition comprising an anti-CD79b immunoconjugate at a concentration of about 10 mg / mL to about 20 mg / mL, a surfactant at a concentration of at least 0.06% w / v (i.e., 0.6 mg / mL), a succinate buffer at a concentration of about 10 mM to about 200 mM, and a sugar at a concentration of about 100 mM to about 260 mM, wherein the liquid pharmaceutical composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0306]

[0307] in:

[0308] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (such as between about 2 and about 5). Between, for example, approximately 3.5).

[0309] In some embodiments, the pharmaceutical composition is reconstituted in SWFI (1:1 mass:volume). In some embodiments, the pharmaceutical composition is reconstituted in water to form a liquid pharmaceutical composition comprising 20 mg / mL anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v polysorbate 20, and 120 mM sucrose, wherein the liquid pharmaceutical composition has a pH of 5.3.

[0310] In some embodiments, the pharmaceutical composition is reconstituted in SWFI and then diluted in isotonic buffer in an IV infusion bag, wherein the concentration of the diluted surfactant in the IV infusion bag is at least 0.003% w / v. In one embodiment, the concentration of the diluted surfactant in the IV infusion bag is at least 0.004% w / v. In one exemplary embodiment, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one specific embodiment, the concentration of sucrose is 120 mM. In some embodiments, the succinate buffer is sodium succinate buffer. In one specific exemplary embodiment, the concentration of sodium succinate buffer is 10 mM.

[0311] In some embodiments, the stability of the pharmaceutical compositions described herein after reconstitution in water is assessed by aggregation, pH, turbidity, the number of visible and / or invisible particles, or the content of free (e.g., unconjugated) drug. In some embodiments, stability may be assessed using ultraviolet spectrophotometry, SE-HPLC, hydrophobic interaction chromatography (HIC), icIEF, or HIAC, or combinations thereof. In some embodiments, the pharmaceutical compositions remain stable at 30°C for a maximum of about 1 day, a maximum of about 2 days, or a maximum of about 3 days after reconstitution in water. In some embodiments, the pharmaceutical compositions remain stable at 5°C ± 3°C for a maximum of about 1 day, a maximum of about 2 days, a maximum of about 3 days, a maximum of about 4 days, a maximum of about 5 days, a maximum of 6 days, or a maximum of about 7 days after reconstitution in water.

[0312] In some embodiments, the stability of the composition redissolved in water was determined by size exclusion high-performance liquid chromatography (SE-HPLC). In one exemplary embodiment, the composition has a main peak (peak area %) of at least 95.0 as determined by SE-HPLC.

[0313] In some specific embodiments, the composition has an HMWS (%) of less than 4.0 as measured by SE-HPLC. In some specific embodiments, the composition has an LMWS (%) of less than 1.0 as measured by SE-HPLC. In some embodiments, the composition has a main peak (peak area %) of at least 95.0 and an HMWS of less than 4.0 as measured by SE-HPLC. In some embodiments, the composition has a main peak (peak area %) of at least 95.0, an HMWS (%) of less than 4.0, and an LMWS (%) of less than 1.0 as measured by SE-HPLC.

[0314] In some embodiments, the stability of the composition redissolved in water is determined by imaging capillary isoelectric focusing (icIEF). In one embodiment, the composition has a main peak (peak area %) of at least 73.0, an acidic region (peak area %) of up to 25.5, and an alkaline region (peak area %) of up to 10.0, as measured by icIEF. In an exemplary embodiment, the composition has a main peak (peak area %) of at least 58.0, an acidic region (peak area %) of up to 32.0, and an alkaline region (peak area %) of up to 12.0, as measured by icIEF.

[0315] In some embodiments, the reconstituted pharmaceutical composition comprises 20 mg / mL polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, and 1.2 mg / mL polysorbate 20, and remains chemically stable after storage at pH 5.3 at a temperature between about 2°C and about 8°C for 72 hours. In some embodiments, the reconstituted pharmaceutical composition is stably stored at a temperature between about 2°C and about 8°C for 72 hours without reducing the bioactivity of polatuzumab vedotin. In some embodiments, the reconstituted pharmaceutical composition comprises 20 mg / mL polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, and 1.2 mg / mL polysorbate 20, and remains chemically stable after storage at pH 5.3 at 30°C for 24 hours under exposure to ambient light. In some embodiments, the reconstituted pharmaceutical composition does not reduce the bioactivity of polatuzumab vedotin after being stored at 30°C for 24 hours under conditions of exposure to ambient light.

[0316] In one aspect, this disclosure provides a liquid composition comprising an anti-CD79b immunoconjugate, a surfactant, a succinate buffer, and a sugar, wherein the concentration of the anti-CD79b immunoconjugate is from about 10 mg / mL to about 20 mg / mL, the concentration of the surfactant is at least 0.06% w / v (i.e., 0.6 mg / mL), the concentration of the succinate buffer is from about 10 mM to about 200 mM, and the concentration of the sugar is from about 100 mM to about 260 mM, wherein the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0317]

[0318] in:

[0319] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and the p value is from about 1 to about 8 (e.g., between about 2 and about 5). Between, for example, approximately 3.5).

[0320] In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one exemplary embodiment, the concentration of sucrose is 120 mM. In some embodiments, the succinate buffer is sodium succinate buffer. In one specific exemplary embodiment, the concentration of sodium succinate buffer is 10 mM.

[0321] In some embodiments, the liquid composition of this disclosure is dissolved in a buffer solution. The buffer solution may be, but is not limited to, physiological saline (0.9% w / v sodium chloride), hemisaline (0.45% w / v sodium chloride), 5% w / v glucose, lactated Ringer's solution, or combinations thereof.

[0322] In some embodiments, the liquid composition of this disclosure is dissolved in isotonic buffer. In some embodiments, the isotonic buffer may be prepared fresh or packaged in a pre-filled IV infusion bag. In some embodiments, the isotonic buffer is physiological saline (0.9% sodium chloride). In one exemplary embodiment, the liquid composition of this disclosure is dissolved in isotonic buffer in an IV infusion bag.

[0323] In some embodiments, the liquid composition of this disclosure is dissolved in an isotonic buffer solution in an IV infusion bag having a pH of about 4.8 to about 5.8. In one specific embodiment, the pH is about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, or about 5.8. In some embodiments, the liquid composition of this disclosure is dissolved in an isotonic buffer solution in an IV infusion bag having a pH of about 5.3.

[0324] In some embodiments, the stability of the liquid composition in the IV infusion bag described herein is measured by aggregation, pH, turbidity, the number of visible and / or invisible particles, or the content of free (e.g., unconjugated) drug. In some embodiments, the stability of the liquid composition in the IV infusion bag may be evaluated using ultraviolet spectrophotometry, SE-HPLC, hydrophobic interaction chromatography (HIC), icIEF, or HIAC, or combinations thereof.

[0325] In some embodiments, the liquid composition in the IV infusion bag of this disclosure remains stable under static conditions. In some embodiments, the liquid composition in the IV infusion bag of this disclosure remains stable under stirring conditions. In some embodiments, the liquid composition in the isotonic buffer solution of the IV infusion bag of this disclosure remains stable at 30°C for a maximum of about 1 hour, a maximum of about 2 hours, a maximum of about 3 hours, a maximum of about 4 hours, a maximum of about 5 hours, a maximum of about 6 hours, a maximum of about 7 hours, or a maximum of about 8 hours. In some embodiments, the liquid composition in the isotonic buffer solution of the IV infusion bag of this disclosure remains stable at 25°C for a maximum of about 8 hours, a maximum of about 9 hours, a maximum of about 10 hours, a maximum of about 12 hours, a maximum of about 14 hours, a maximum of about 16 hours, a maximum of about 18 hours, a maximum of about 20 hours, a maximum of about 22 hours, or a maximum of about 24 hours. In some embodiments, the liquid composition in the isotonic buffer solution dissolved in an IV infusion bag as described herein remains stable at 5°C ± 3°C for a maximum of about 24 hours, a maximum of about 30 hours, a maximum of about 36 hours, a maximum of about 42 hours, a maximum of about 48 hours, a maximum of about 54 hours, a maximum of about 60 hours, a maximum of about 66 hours, or a maximum of about 72 hours. In some embodiments, the isotonic buffer solution is physiological saline.

[0326] In some embodiments, the reconstituted or dilute solutions for infusion described herein (e.g., dilute solutions for infusion containing immunoconjugates (e.g., polatuzumab vedotin)) are sterile solutions.

[0327] III. Treatment Methods

[0328] The pharmaceutical or liquid compositions described in this disclosure are envisioned for use in treating a variety of proliferative disorders characterized by the overexpression of tumor antigens. One exemplary proliferative disorder is cancer. Examples of cancer include, but are not limited to, hematopoietic system cancers or blood-related cancers such as lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as splenic cancer and lymph node cancer, and malignant tumors, blastomas, and sarcomas. More specific examples of cancer include B-cell proliferative disorders, such as high, intermediate, and low-grade lymphomas (including B-cell lymphomas, such as mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin lymphoma (NHL), mantle cell lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, and T-cell lymphoma) and leukemias (including secondary leukemia, chronic lymphocytic leukemia (CLL) such as B-cell leukemia (CD5+ B lymphocytes), myeloid leukemia such as acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia such as allergic lymphocytic leukemia (ALL), and spinal dysplasia), as well as other cancers associated with blood and / or B-cells or T-cells. Cancers also include other hematopoietic cells, including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils, and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells. In some embodiments, the proliferative disorders described herein are recurrent or refractory cancers.

[0329] In some embodiments, the pharmaceutical compositions or liquid compositions described herein may be used to treat relapsed leukemia or relapsed lymphoma. In other specific embodiments, the pharmaceutical compositions or liquid compositions described herein may be used to treat refractory leukemia or refractory lymphoma.

[0330] In one aspect, this disclosure provides a method for treating a proliferative disorder in a patient in need, the method comprising administering to the patient a pharmaceutical composition or liquid composition described herein.

[0331] In some embodiments, the cancer to be treated is a B-cell proliferative disorder. In specific embodiments, the B-cell proliferative disorder is selected from the group consisting of: lymphoma, myeloma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), aggressive NHL, indolent lymphoma, follicular lymphoma (FL), relapsed aggressive NHL, relapsed indolent NHL, relapsed NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma.

[0332] In one exemplary embodiment of this disclosure, the B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL). In one exemplary embodiment, the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL). In one specific embodiment, the B-cell proliferative disorder is relapsed diffuse large B-cell lymphoma (DLBCL). In another specific embodiment, the B-cell proliferative disorder is refractory diffuse large B-cell lymphoma (DLBCL).

[0333] In one exemplary embodiment, the B-cell proliferative disorder is relapsed or refractory NHL. In yet another exemplary embodiment, the B-cell proliferative disorder is follicular lymphoma.

[0334] The cancer may contain cells expressing CD79b, enabling the anti-CD79b immunoconjugates described herein to bind to cancer cells. Various diagnostic / prognostic assays can be used to determine CD79b expression in cancer. In one embodiment, CD79b overexpression can be analyzed by IHC. IHC analysis can be performed on paraffin-embedded tissue sections from tumor biopsies, using CD79b protein staining intensity criteria related to staining degree and the proportion of tumor cells examined.

[0335] For prevention or treatment of a condition, the appropriate ADC dose depends on the type of disease to be treated (as defined above), the severity and duration of the disease, whether the molecule is administered for prophylactic or therapeutic purposes, the patient's medical history and response to antibodies, and the attending physician's discretion. The molecule is administered to the patient either once or as part of a series of treatments. Depending on the type and severity of the disease, a molecule of about 1 µg / kg to 15 mg / kg (e.g., 0.1–20 mg / kg) may be a candidate initial dose for administration to the patient, whether, for example, by a single or multiple administrations or by continuous infusion. Depending on the factors above, a typical daily dose range can be from about 1 µg / kg to 100 mg / kg or more. An exemplary dose of ADC administered to a patient is in the range of about 0.1 to about 10 mg / kg of the patient's body weight. For repeated administration over several days or longer, depending on the condition, treatment continues until the desired suppression of disease symptoms occurs.

[0336] For the treatment or prevention of proliferative disorders, the pharmaceutical composition or liquid composition described herein is administered via intravenous infusion. The dosage range for infusion is approximately 1 µg / m² per dose. 2 Approximately 10,000 µg / m 2It is usually administered once a week, for a total of one, two, three, or four doses. Alternatively, the dosage range is approximately 1 µg / m². 2 Approximately 1000 µg / m 2 Approximately 1 µg / m 2 Approximately 800 µg / m 2 Approximately 1 µg / m 2 Approximately 600 µg / m 2 Approximately 1 µg / m 2 Approximately 400 µg / m 2 Approximately 10 µg / m 2 Approximately 500 µg / m 2 Approximately 10 µg / m 2 Approximately 300 µg / m 2 Approximately 10 µg / m 2 Approximately 200 µg / m 2 or approximately 1 µg / m 2 Approximately 200 µg / m 2 The administration frequency may be once daily, once weekly, multiple times weekly but less than once daily, multiple times monthly but less than once daily, multiple times monthly but less than once weekly, once monthly, or intermittently, to relieve or reduce the symptoms of the disease. Administration may continue at any interval disclosed herein until the symptoms of the tumor or the treated lymphoma or leukemia are relieved. Once symptom relief or reduction is achieved, administration may be continued if such relief or reduction can be prolonged by continuing administration.

[0337] Various detection methods can be used to determine CD79b expression in cancer. In one embodiment, CD79b peptide overexpression can be analyzed by immunohistochemistry (IHC). IHC analysis can be performed on paraffin-embedded tissue sections from tumor biopsies, using the following CD79b protein staining intensity criteria:

[0338] 0 points - No staining or membrane staining was observed in less than 10% of tumor cells.

[0339] 1+ - Weak / virtually imperceptible membrane staining was detected in more than 10% of tumor cells. Only a portion of the membrane of these cells was stained.

[0340] 2+ points - Weak to moderate intact membrane staining was observed in more than 10% of tumor cells.

[0341] 3+ points - Moderate to strong intact membrane staining was observed in more than 10% of tumor cells.

[0342] Tumors with a CD79b peptide expression score of 0 or 1+ may be characterized by not overexpressing CD79b, while tumors with a score of 2+ or 3+ may be characterized by overexpressing CD79b.

[0343] Alternatively or additionally, formalin-fixed, paraffin-embedded tumor tissue can be subjected to FISH assays, such as INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois), to determine CD79b overexpression in the tumor.

[0344] CD79b overexpression or amplification can be assessed by in vivo assays, for example, by administering a molecule (e.g., an antibody) that binds to the molecule to be detected and carries a detectable label (e.g., a radioactive isotope or a fluorescent label) and scanning the patient from outside to locate the label.

[0345] combination therapy

[0346] Currently, cancer treatment, depending on the stage of cancer, involves one or more of the following therapies: surgical removal of cancerous tissue, radiotherapy, and chemotherapy. Anti-CD79b immunoconjugate therapy is particularly suitable for elderly patients who cannot well tolerate the toxicity and side effects of chemotherapy, and for metastatic diseases with limited response to radiotherapy. Tumors targeting anti-CD79b immunoconjugates as described in this disclosure can be used to alleviate CD79b-expressing cancers during initial diagnosis or recurrence of the disease. For therapeutic application, anti-CD79b immunoconjugates can be used alone or in combination with, for example, hormones, anti-angiogenic agents, or radiolabeled compounds, or in combination with surgery, cryotherapy, and / or radiotherapy. The administration of the pharmaceutical compositions or liquid compositions described in this disclosure can be combined with other forms of conventional treatment, or administered continuously before or after conventional treatment. Chemotherapy drugs such as TAXOTERE® (docetaxel), TAXOL® (paclitaxel), estradiol, and mitoxantrone are used to treat cancer, particularly in high-risk patients. In the methods for treating or alleviating cancer described in this disclosure, anti-CD79b antibodies may be administered to cancer patients in combination with one or more of the aforementioned chemotherapeutic agents. Specifically, combination therapy with paclitaxel and modified derivatives is conceivable (see, for example, EP0600517). The pharmaceutical compositions or liquid compositions described in this disclosure may be administered together with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, the pharmaceutical compositions or liquid compositions described in this disclosure may be administered in combination with chemotherapy to enhance the activity and efficacy of chemotherapeutic agents such as paclitaxel. The Physician's Desk Reference (PDR) discloses dosages of these drugs already used to treat various cancers. The therapeutically effective dosing regimens and dosages of these aforementioned chemotherapeutic drugs will depend on the specific cancer being treated, the severity of the disease, and other factors familiar to those skilled in the art, and may be determined by a physician.

[0347] In another embodiment, the pharmaceutical or liquid compositions described in this disclosure may be administered in combination with one or more chemotherapeutic agents or growth inhibitors, including in combination with mixtures of different chemotherapeutic agents or other cytotoxic agents or other therapeutic agents that also inhibit tumor growth. Chemotherapy agents include estradiol phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea, and hydroxyurea-paclitaxel (such as paclitaxel and docetaxel) and / or anthracycline antibiotics. The preparation and dosing schedule of such chemotherapeutic agents may be determined according to the manufacturer's instructions or by the experience of a skilled practitioner. The preparation and dosing schedule of such chemotherapeutic agents are also described in: Chemotherapy Service Ed., MCPerry, Williams & Wilkins, Baltimore, MD (1992). The pharmaceutical or liquid compositions described in this disclosure may be administered in combination with the following drugs at known doses: antihormonal compounds (e.g., antiestrogenic compounds such as tamoxifen); antiprogesterone compounds such as onaphalistatin (see EP 616 812); or antiandrogens such as flutamide. In cases where the cancer to be treated is androgen-independent, the patient may have previously received anti-androgen therapy, and the pharmaceutical or liquid compositions described herein may be administered to the patient after the cancer becomes androgen-independent.

[0348] Sometimes, it may be beneficial to administer cardioprotective agents (for the prevention or reduction of treatment-related myocardial dysfunction) or one or more cytokines to patients in combination. In addition to the treatment regimens described above, patients may undergo surgical resection of cancer cells and / or receive radiation therapy (e.g., external beam irradiation or radiolabeled agents such as antibody therapy) before, during, or after receiving the pharmaceutical or liquid compositions described in this disclosure. Appropriate doses of any of the aforementioned combined-administered drugs are currently used doses, and their doses may be reduced due to the combined effect (synergistic effect) of the drug with the anti-CD79b antibody.

[0349] In some embodiments, the pharmaceutical or liquid compositions described herein may be administered to a patient in combination with an effective amount of another therapeutic agent. In one embodiment, the therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, cytotoxic agents, anti-angiogenic agents, immunosuppressants, prodrugs, cytokines, cytokine antagonists, cytotoxic radiotherapy, corticosteroids, anticancer vaccines, and growth inhibitors. In another embodiment, the therapeutic agent is selected from tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab, vincristine, imatinib mesylate, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, methotrexate, vincristine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, bortezomib, melphalan, prednisone, prednisolone, and docetaxel.

[0350] In some embodiments, the therapeutic agent is an anti-CD20 antibody.

[0351] In some embodiments, the pharmaceutical or liquid compositions described herein may be administered in combination with an effective amount of an anti-CD20 antibody for the treatment of B-cell proliferative disorders. In one embodiment, the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL). In another embodiment, the proliferative disorder is follicular lymphoma (FL). In some embodiments, the anti-CD20 antibody is rituximab.

[0352] In some embodiments, the therapeutic agent is a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent includes one or more of cyclophosphamide, daunorubicin, vincristine, and prednisone. In another embodiment, the chemotherapeutic agent includes cyclophosphamide, doxorubicin, and prednisone.

[0353] In some embodiments, the pharmaceutical or liquid compositions described herein may be administered in combination with an effective amount of an anti-CD20 antibody and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent includes cyclophosphamide, doxorubicin, and prednisone.

[0354] In some embodiments, the pharmaceutical or liquid compositions described herein may be administered in combination with an effective amount of an anti-CD20 antibody and a chemotherapeutic agent for treating B-cell proliferative disorders. In one embodiment, the B-cell proliferative disorder is non-Hodgkin lymphoma (NHL). In another embodiment, the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL). In one specific embodiment, DLBCL is relapsed / refractory DLBCL. In another embodiment, NHL is relapsed NHL or refractory NHL. In another specific embodiment, the proliferative disorder is follicular lymphoma (FL). In another embodiment, FL is relapsed / refractory FL. In some embodiments, the anti-CD20 antibody is rituximab.

[0355] In some embodiments, the pharmaceutical or liquid compositions described herein may be administered in combination with an anti-CD20 antibody (naked antibody or ADC). In one embodiment, the anti-CD20 antibody is rituximab (Rituxan®) or a biosimilar thereof. In some embodiments, the anti-CD20 antibody is ocrelizumab (2H7) (Genentech, Inc., South San Francisco, CA) or a biosimilar thereof. In another embodiment, the pharmaceutical or liquid compositions described herein may be administered in combination with an anti-VEGF antibody (e.g., Avastin®).

[0356] In some embodiments, the pharmaceutical or liquid compositions described herein may be administered in combination with an alkylating agent. In some embodiments, the pharmaceutical or liquid compositions described herein may be administered in combination with an alkylating agent and an anti-CD20 antibody (naked antibody or ADC). In some embodiments, the anti-CD20 antibody is rituximab (Rituxan®) or a biosimilar thereof. In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butyric acid and its salts. In some embodiments, the alkylating agent is bendamustine.

[0357] Combination therapy can be administered simultaneously or sequentially. When administered sequentially, the composition may be administered two or more times. Combination administration includes the combined administration of individual formulations or single-drug formulations, as well as sequential administration in any order, wherein preferably, both (or all) of the active agents exert their biological activity simultaneously over a period of time.

[0358] The appropriate dose of any of the above-mentioned combination drugs is the currently used dose, and its dose may be reduced due to the combined effect (synergistic effect) of newly identified drugs with other chemotherapeutic agents or treatments.

[0359] Combination therapy can provide a “synergistic effect” and is demonstrated to be “synergistic,” meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects of using the compounds alone. A synergistic effect can be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage form; (2) administered alternately or as separate formulations in parallel; or (3) administered via certain other regimens. When delivered using alternating therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example by injection into separate syringes. Generally, in alternating therapy, various active ingredients are administered sequentially, i.e., continuously, in effective doses, while in combination therapy, two or more active ingredients are administered together in effective doses.

[0360] IV. Products and Reagent Kits

[0361] Another embodiment of this disclosure is an article comprising materials that can be used to treat, prevent, and / or diagnose proliferative disorders. In one embodiment, the article comprises a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition for the effective treatment, prevention, and / or diagnosis of cancerous conditions, and the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an anti-CD79b immunoconjugate as described in this disclosure. The label or packaging insert indicates that the composition is for the treatment of cancer. The label or packaging insert will further contain instructions for administering the composition to a cancer patient. Additionally, the article may further comprise a second container comprising pharmaceutically acceptable buffers, such as sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The product may also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0362] In some embodiments, the article of manufacture is a container such as a bottle, vial, or syringe containing the pharmaceutical composition described herein. In some embodiments, the article of manufacture is a container such as a bottle, vial, or syringe containing the lyophilized pharmaceutical composition (such as a block) described herein. In some embodiments, the article of manufacture is a container such as a bottle, vial, or syringe containing the reconstituted lyophilized pharmaceutical composition described herein. In one specific embodiment, the article of manufacture is a container containing the lyophilized pharmaceutical composition reconstituted in sterile water for injection (SWFI).

[0363] In some embodiments, the article is a plastic container containing the pharmaceutical composition described herein. In some embodiments, the article is a glass container containing the pharmaceutical composition described herein.

[0364] In some embodiments, the article is a glass vial containing the pharmaceutical composition described in this disclosure. In one specific embodiment, the article is a glass vial containing the lyophilized pharmaceutical composition (such as a block) described in this disclosure. In one specific embodiment, the article is a glass vial containing the reconstituted lyophilized pharmaceutical composition (such as a block) described in this disclosure. In one specific embodiment, the article is a container containing the lyophilized pharmaceutical composition (such as a block) reconstituted in sterile water for injection (SWFI).

[0365] In some embodiments, the article is an IV infusion bag comprising the pharmaceutical composition or liquid composition described in this disclosure. The IV infusion bag may be composed of materials including, but not limited to, polyolefin (PO), polyvinyl chloride (PVC), ethylene vinyl acetate, polypropylene (PP), polyethylene (PE), copolyester ether, or combinations thereof. In some embodiments, the IV infusion bag is a polyolefin (PO), polypropylene (PP), polyethylene (PE), or polyvinyl chloride (PVC) bag.

[0366] In some embodiments, an article comprising a pharmaceutical composition or liquid composition of the present disclosure may contain at least about 5 mL, at least about 10 mL, at least about 15 mL, at least about 20 mL, at least about 25 mL, at least about 30 mL, at least about 35 mL, at least about 40 mL, at least about 45 mL, at least about 50 mL, at least about 55 mL, at least about 60 mL, at least about 65 mL, at least about 70 mL, at least about 75 mL, at least about 80 mL, at least about 85 mL, at least about 90 mL, at least about 95 mL, at least about 100 mL, at least about 105 mL, at least about 110 mL, at least about 115 mL, at least about 120 mL, at least about 125 mL, at least about 130 mL, at least about 135 mL, at least about 140 mL, at least about 145 mL, at least about 150 mL, at least about 155 mL, at least about 160 mL, at least about 165 mL, at least about 170 mL, at least about 175 mL. The article has a volume of at least about 180 mL, at least about 185 mL, at least about 190 mL, at least about 195 mL, or at least about 200 mL. In some embodiments, the article is a PO bag that can hold a volume of at least about 25 mL, at least about 50 mL, or at least about 100 mL. In some embodiments, the article is a PVC bag that can hold a volume of at least about 25 mL, at least about 50 mL, or at least about 100 mL.

[0367] In some embodiments, the article of manufacture is an IV infusion bag comprising the pharmaceutical composition or liquid composition described herein and a buffer solution. In one specific embodiment, the buffer solution may be physiological saline, semi-saline, 5% w / v glucose, lactated Ringer's solution, or a combination thereof. In one embodiment, the article of manufacture is an IV infusion bag comprising the pharmaceutical composition or liquid composition described herein dissolved in semi-saline.

[0368] In some embodiments, the article of manufacture is an IV infusion bag comprising the pharmaceutical composition or liquid composition described herein and an isotonic buffer solution. In one embodiment, the article of manufacture is an IV infusion bag comprising the pharmaceutical composition or liquid composition described herein dissolved in physiological saline.

[0369] In one exemplary embodiment, the article is a PVC bag containing the pharmaceutical composition or liquid composition described in this disclosure and physiological saline, which can hold a volume of at least about 100 mL. In another exemplary embodiment, the article is a PVC bag containing the pharmaceutical composition or liquid composition described in this disclosure and physiological saline, which can hold a volume of about 100 mL.

[0370] In another exemplary embodiment, the article is a PO bag containing the pharmaceutical composition or liquid composition described in this disclosure and physiological saline, which can hold a volume of at least about 100 mL. In yet another exemplary embodiment, the article is a PO bag containing the pharmaceutical composition or liquid composition described in this disclosure and semi-saline, which can hold a volume of at least about 100 mL.

[0371] In any of the embodiments herein, a stable pharmaceutical or liquid pharmaceutical composition may be stored in a container such as a bottle, vial, syringe, or intravenous (IV) infusion bag.

[0372] Kits are also provided for use for various purposes, such as cell-killing assays expressing CD79b. Like the products, the kits include a container and a label or packaging insert on or associated with the container. The container contains a composition comprising at least one anti-CD79b immunoconjugate described herein. Additional containers may be included, containing, for example, diluents, surfactants, buffers, or control antibodies. The label or packaging insert provides a description of the composition and instructions for its intended use.

[0373] The following description is provided to enable those skilled in the art to prepare and use various embodiments. The descriptions of specific devices, techniques, and applications are provided by way of example only. Various modifications to the embodiments described herein will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other instances and applications without departing from the spirit and scope of the various embodiments described. Therefore, the various embodiments described are not intended to be limited to the examples described and illustrated herein, but are consistent with the scope of the claims.

[0374] Example

[0375] Example 1: Study on surfactants for anti-CD79b immunoconjugates

[0376] The formulation initially used in early clinical trials of anti-CD79b-vc-MMAE in patients was a liquid formulation containing histidine acetate buffer and 0.02% polysorbate 20, delivered to patients via an intravenous infusion pump. To determine the commercial formulation of the drug, various nonionic surfactants at different concentrations were tested to determine whether they could protect and stabilize anti-CD79b-vc-MMAE against aggregation after dilution in IV infusion bags, as intravenous infusion was the route of delivery to patients in a commercial setting. Specifically, the stability of anti-CD79b-vc-MMAE was evaluated under (a) static conditions in IV infusion bags and (b) air-water interfacial stress conditions induced by short-term agitation in IV infusion bags.

[0377] Anti-CD79b-vc-MMAE

[0378] Anti-CD79b-vc-MMAE was diluted with 20 mM histidine acetate (pH 5.5). A 20 mM histidine acetate pH 5.5 buffer was prepared by adding 3.11 g histidine and 1.03 mL glacial acetic acid to 800 mL of fresh ultrapure water. The measured pH was 5.5 ± 0.1. The resulting solution was brought to a final volume of 1 L with fresh ultrapure water, filtered through a 0.22 µMPES filter, and stored at 2–8 °C. Anti-CD79b-vc-MMAE was tested at the following concentrations: (i) 10 mg / mL, and (ii) 20 mg / mL (diluted to 10 mg / mL or 20 mg / mL with the filtered buffer as needed). The diluted substances were stored protected from light at 2–8 °C.

[0379] surfactants

[0380] The surfactants tested were: (i) polysorbate 20 (PS20), USP NF grade, manufactured by Croda; (ii) polysorbate 80 (PS80), USP NF grade, manufactured by Croda; (iii) poloxamer (P188), USP NF grade, containing 98 ppm butylated hydroxytoluene, manufactured by Spectrum Chemicals; and (iv) N-octyl-β-D-glucopyranoside (OG) C14H28O6, FW: 292.4, manufactured by Affymetrix, analytical grade. The 10%, 1.0%, and 0.5% PS20 stock solutions were prepared as follows: 10%, 1%, or 0.5% PS20 stock solutions prepared in ultrapure water were used, depending on the target concentration of PS20 in the bag. A 10% PS20 stock solution was prepared by adding 10 g of PS20 to a 100 mL volumetric flask. 60 mL of ultrapure water was added to the flask, and the mixture was stirred with a magnetic stirrer to ensure thorough mixing. After allowing the bubbles to dissipate for 30 minutes, the flask was filled to 100 mL with ultrapure water. The solution was then transferred to a light-protected container and stored at 2–8°C for up to one week. A 1.0% PS20 solution was prepared using a similar procedure with 1.0 g of PS20 in 100 mL of ultrapure water. A 0.5% PS20 solution was prepared using a 1:1 (v / v) dilution of the 1.0% PS20 stock solution.

[0381] The preparation method for 0.5% PS80 is as follows: A 10% PS80 solution is prepared by adding 10 g of PS80 to a 100 mL volumetric flask. Add 50 mL of ultrapure water to the flask and stir with a magnetic stirrer until the solution is thoroughly mixed. Let the solution stand for 30 minutes to allow air bubbles to dissipate, then remove the stir bar. Then, bring the volume of the solution to 100 mL using ultrapure water. To obtain a 0.5% PS80 stock solution, bring 0.5 mL of the 10% PS80 stock solution to 10 mL using ultrapure water. Transfer the solution to a light-protected container and store at 2–8°C for up to one week.

[0382] The preparation method of 0.5% P188 is as follows: A 10% P188 solution is prepared by adding 10 g of P188 to a 100 mL volumetric flask. 60 mL of ultrapure water is added to the flask, and the mixture is stirred with a magnetic stirrer until fully mixed. The solution is allowed to stand for 30 minutes to allow air bubbles to dissipate, then the stir bar is removed. The solution is then brought to a final volume of 100 mL with ultrapure water. To obtain a 0.5% P188 stock solution, 0.5 mL of the 10% P188 stock solution is brought to a final volume of 10 mL with ultrapure water. The solution is transferred to a light-protected container and stored at 2–8°C for up to one week.

[0383] A. Study on static IV infusion bags:

[0384] Dilute the anti-CD79b-vc-MMAE into an IV infusion bag. To mimic the pharmacy-recommended preparation procedure, remove an equal volume of saline from the IV infusion bag to the total volume of anti-CD79b-vc-MMAE and surfactant to be added. Introduce the surfactant into the IV infusion bag before adding the anti-CD79b-vc-MMAE formulation in histidine buffer to ensure that the anti-CD79b-vc-MMAE, acting as a surfactant-free ultrafiltration percolation (UFDF) chamber, is not exposed to any surfactant-free saline in the IV infusion bag. Then gently rotate the IV infusion bag to ensure complete mixing, avoiding vigorous shaking or agitation of the bag containing the anti-CD79b-vc-MMAE.

[0385] Anti-CD79b-vc-MMAE was prepared in a glass vial containing saline solution: various amounts of saline solution (0.9% sodium chloride), surfactant, and immunoconjugate were added to a 15 cc Forma Vitrum glass vial, which was then sealed with a 20 mm Daikyo stopper. The samples were subsequently incubated under specific research conditions.

[0386] Sample collection: Throughout the study, samples were transferred using a 1 cc or 5 cc BD Falcon syringe with an 18 G needle. The collected samples were stored in a 10 cc PETG (Nalgene) container.

[0387] Protein concentration was determined using ultraviolet spectrophotometry: The protein concentration of the selected sample after dilution was determined using an Agilent spectrophotometer. Samples collected from IV infusion bags were volumetrically diluted (if necessary) to bring the UV signal between 0.1 and 1.0 AU. Absorbance was recorded at 279 nm and 320 nm. The UV concentration was calculated using an extinction coefficient of 1.40 (mg / mL)⁻¹ cm⁻¹. The corrected A279 was obtained by subtracting A320 nm from A279 nm. This correction addressed the effect of solution turbidity, enabling accurate measurement of protein concentration.

[0388] Turbidity: The turbidity of the sample was measured by recording the average UV absorbance at 340–360 nm using an Agilent spectrophotometer equipped with a 1 cm path length cuvette. Purified water was used as a blank for the spectrophotometer.

[0389] Visible Particles: The presence of visible particles in the sample within a 10 cc PETG container was visually assessed using a black and white lightbox. Soluble aggregate content (as an indicator of protein instability) was determined using an Agilent 1200 / 1280 high-performance size exclusion chromatography (HPSEC) (Agilent Technologies, Santa Clara, California) equipped with a diode array detector (set to 280 nm) and a Tosoh Bioscience LLC (Montgomeryville, Pennsylvania) TSK-Gel G3000SWXL size exclusion column (300 × 7.8 mm, 5 µm) (column temperature at ambient temperature). The sample was eluted with 0.2 M K₃PO₄ and 0.25 M KCl (pH 6.2) at an isocratic flow rate of 0.5 mL / min over 30 min.

[0390] High-precision liquid particle counter (HIAC): The HIAC is used for particle size determination and counting in solution. Using a HIAC (model 9703+), visible and / or invisible particles (SVP) were quantitatively determined by optical obscuration. The following particle sizes were collected: 1.6, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, and 50 µm.

[0391] Stability study of IV bags in static storage:

[0392] Preliminary studies of surfactant in IV infusion bags were conducted under both static and agitated conditions. The dose range of the anti-CD79b-vc-MMAE used in these studies was designed to be 1.8 mg / kg to 2.4 mg / kg and was maintained within this range. It was assumed that most patients weighed between 40 kg and 120 kg. Based on these assumptions, the total dose per IV infusion bag varied between 72 mg (low dose / low body weight) and 288 mg (high dose / high body weight).

[0393] Figures 1 and 2 summarize the performance results of the IV infusion bag, with a fixed PS20 concentration of 0.005% and a final anti-CD79b-vc-MMAE concentration of 0.65 mg / mL. Assuming a minimum patient weight of 40 kg, a low dose of 1.8 mg / kg was used in a 100 mL IV infusion bag to determine the optimal concentration of anti-CD79b-vc-MMAE. Since the overfill volume of the 100 mL IV infusion bag is 10 mL, the final concentration in the IV infusion bag is: (1.8 mg / kg × 40 kg) / 110 mL = 0.65 mg / mL.

[0394] After static storage at 30°C for 22 hours, there was no significant difference in the physical stability of the IV infusion bag type against CD79b-vc-MMAE. Regarding high molecular weight substances (HMWS) as measured by SEC, all bags showed an increase of 0.3% ± 0.1% (∆). This indicates that the anti-CD79b-vc-MMAE is unstable at 30°C after dilution with 0.9% NaCl.

[0395] Surfactants and screening studies under static storage conditions:

[0396] To ensure adequate control and efficient screening of surfactant types and concentrations, a fixed protein concentration must be maintained within the bag. The concentration of anti-CD79b-vc-MMAE in the bag was 2.6 mg / mL, selected based on the maximum possible dose of 2.4 mg / kg for a patient weighing up to 120 kg, administered via a 100 mL bag. This bag size is recommended for clinical use and allows for a 10% overfill volume, as calculated below: (2.4 mg / kg × 120 kg) / 110 mL = 2.6 mg / mL. The highest protein concentration in the bag is assumed to be the worst-case scenario, as higher bulk protein concentrations would encourage faster antibody adsorption to the air-water interface, leading to higher levels of interface-mediated aggregation.

[0397] The effect of temperature on stability under static storage conditions:

[0398] Providing appropriate storage conditions for combined IV infusion bags is crucial for the product quality of antibody therapeutics, and these storage conditions were evaluated. This information is also needed in the preparation of pharmacy manuals and product instructions to ensure the correct use of any antibody therapeutic. Various concentrations of PS20 in the bags were tested at temperatures of 30°C, 25°C, and 2–8°C. At 2–8°C, no significant change in HMWS was observed after 22 hours of static storage for the tested PS20 concentrations (0.003%, 0.005%, and 0.01%) in the bags (Figure 3). All changes (% HMWS) were < 0.02%. At 25°C and with PS20 concentrations of 0.005%, 0.01%, 0.015%, 0.025%, and 0.030%, respectively, an increase in HMWS of approximately 0.1% was observed after 22 hours of static storage (Figure 3). The increase in HMWS at 30°C was significantly greater than the increase observed at 25°C. With PS20 content in the bag being 0.004%, 0.015%, 0.02%, 0.025%, 0.05%, and 0.1%, respectively, an increase of 0.3 ± 0.1% in HMWS was observed after 22 hours of static storage (Figure 3).

[0399] At all temperatures, the increase in HMWS was independent of the PS20 concentration in the bag, indicating that aggregation under static conditions was not mediated by interfacial stress. The increase in HMWS was closely related to storage temperature. These results are in good agreement with the findings of a follow-up study conducted by Beckley, N. et al. (Investigation into Temperature-Induced Aggregation of an Antibody Drug Conjugate. Bioconjugate Chemistry, 24, pp: 1674-1683 (2013)), which showed that the formation of HMWS in antibody drug conjugates (ADCs) at higher temperatures is attributed to increased sensitivity of the tertiary structure to thermal stress, possibly due to the presence of the conjugated drug at the interchain cysteine ​​residue near the CH2 domain.

[0400] Test surfactant mixture:

[0401] Poloxamer 188 (P188) is known to reduce the formation of high molecular weight surfactants (HMWS) in IV infusion bags, but invisible particulate matter (SVP) may increase significantly under stirred conditions. In contrast, PS20 does not increase SVP. To further investigate the different results between these two surfactants, a 1:1 weight ratio of PS20 to P188 was used to determine whether PS20 could prevent SVP formation and whether P188 could limit the increase in HMWS. The study was conducted using 25 mL IV infusion bags with an anti-CD79b-vc-MMAE concentration of 2.6 mg / mL. Equal amounts of PS20 and P188 were added on a mass basis to make the total surfactant concentration in the IV infusion bags 0.05% or 0.1%. After static storage at 30°C for 22 hours, an increase in HMWS of 0.3–0.4% was observed, as shown in Table 1 below.

[0402] Table 1: Study of PS20-P188 mixed surfactants under static conditions of 30℃ heat stress

[0403]

[0404] The number of particles larger than 5 µm, 10 µm, and 25 µm increased slightly. When using 0.1% surfactant in IV infusion bags, the number of 2 µm particles increased. Based on these results, the surfactant mixture showed no significant difference compared to PS20 alone.

[0405] This study evaluated four different surfactants, including PS20, PS80, P188, and octyl glucoside (OG). In vials containing saline solution taken from PVC bags, incubation of 0.02% surfactant and 2.6 mg / mL drug at 30°C for 22 hours resulted in an increase in HMWS of 0.3–0.4%, indicating that aggregation was also independent of the type of surfactant used (Figure 4).

[0406] Stability of anti-CD79b-vc-MMAE when stored statically in vials:

[0407] Considering the physical instability of anti-CD79b-vc-MMAE in physiological saline (0.9% NaCl) during static storage in IV infusion bags, the stability of the immunoconjugate in 5% glucose (D5W) and half-saline (0.45% w / v NaCl) vials was tested to assess the dependence of immunoconjugate instability on ionic strength. Solutions containing 0.02% PS20 were prepared, with a fixed concentration of anti-CD79b-vc-MMAE of 2.6 mg / mL. The physical stability of anti-CD79b-vc-MMAE was significantly improved in both half-saline and D5W (Figure 4). In the D5W solution, HMWS remained unchanged, while in the half-saline solution, HMWS increased by no more than 0.1% after static storage at 30°C for 24 hours. Although preparation in D5W limits the aggregation of anti-CD79b-vc-MMAE, antibody glycosylation in D5W solution is a known risk (see Fischer, S. et al., Glycation during Storage and Administration of Monoclonal Antibody Formulations. *European J Pharma and Biopharm*, Vol. 70; pp. 42-50 (2008)). Therefore, due to the possibility of glycosylation, intravenous infusion of anti-CD79b-vc-MMAE in bags containing 5% glucose is no longer considered. Although hemi-saline exhibits a significant improvement in physical stability compared to normal saline, its use for intravenous infusion of anti-CD79b-vc-MMAE is also not preferred due to the low availability of pre-prepared hemi-saline bags in global clinical and commercial settings and the inconvenience and potential risk of microbial contamination associated with preparing hemi-saline bags in pharmacies.

[0408] IV. Conclusions of the static stability study in infusion bags and glass bottles:

[0409] The above static stability studies show that the aggregation of anti-CD79b-vc-MMAE is driven by a high ionic strength environment, indicating that the aggregation rate in the formulation buffer is significantly lower than that in saline.

[0410] B. IV. Study on stirring of infusion bags:

[0411] A series of IV infusion bag agitation studies were conducted to support the transport of composite IV infusion bags containing anti-CD79b-vc-MMAE for drug administration in hospital or clinical settings. Although transporting prepared IV infusion bags containing therapeutic proteins is generally not recommended due to the risk of aggregation, it is frequently necessary in clinical settings, particularly in remote areas with limited pharmacy facilities. Therefore, minimal transport should be supported, and the impact of IV infusion bag agitation on the drug must be evaluated during development. Agitation stress within the IV infusion bag during transport can lead to physical degradation of the antibody therapeutic agent. Physical degradation of the antibody (i.e., aggregation and particle formation) can be mediated by the gas-liquid interface adsorbed into the IV infusion bag and is continuously regenerated during agitation. In the studies described below, an Elliptical Laboratory Shaker (0.75-inch track length) was used for IV infusion bag agitation studies. The IV infusion bag was placed flat on the surface of the shaker.

[0412] The effect of temperature on the stability of IV infusion bags under stirring conditions:

[0413] Agitation studies were conducted on different PS20 concentrations in the bags at 2–8°C, 25°C, and 30°C (Figures 5A to 5C). The rate of increase in HMWS at 30°C was higher than that at 25°C, and also higher than that at 2–8°C. The increase in HMWS was correlated with the temperature in the IV infusion bag during agitation. For bags containing 0.01%, 0.005%, and 0.003% PS20, no increase in HMWS occurred after agitation for 2 hours at 2–8°C (Figure 5A). At 30°C, the increase in HMWS depended on the concentration of PS20 in the bag (Figure 5C). It should be noted that the increase in HMWS was non-linear at all PS20 levels during agitation at 2–8°C (Figure 5A) and 25°C (Figure 5B). This non-linear behavior may be due to sampling at different time points.

[0414] The effect of surfactants on the stability of IV infusion bags under stirred conditions:

[0415] The study used a 1:1 weight ratio of PS20 to P188 mixture to determine whether PS20 could prevent the formation of SVP and whether P188 could limit the increase of HMWS under stirred conditions. The study was conducted using 25 mL IV infusion bags with an anti-CD79b-vc-MMAE concentration of 2.6 mg / mL. Equal amounts of PS20 and P188 were added on a mass basis to achieve a total surfactant concentration of 0.05% or 0.1% in the IV infusion bags.

[0416] When using 0.05% surfactant in IV infusion bags, HMWS increased by 2.9% after stirring at 30°C for 2 hours (Table 2). Particles larger than 5 µm, 10 µm, and 25 µm showed an increasing trend over the longest 1 hour, and the particle count exceeded the limit (LOD) at 2 hours. When using 0.1% surfactant in IV infusion bags, particles of all sizes showed an increasing trend, but the results were significantly lower than those containing 0.05% surfactant (Table 2). Compared to the total surfactant concentration of 0.05% in IV infusion bags, the increase in HMWS after 2 hours of stirring was also reduced by approximately 2.8%. Based on these results, the surfactant mixture was not significantly different from PS20 alone.

[0417] Table 2: Study on PS20-P188 mixed surfactants under heat stress at 30℃ and stirring conditions

[0418]

[0419] Stirring of short-term IV infusion bags at 30°C:

[0420] The aim of this study was to investigate the short-term transport of IV infusion bags containing diluted anti-CD79b-vc-MMAE under ambient conditions and to determine the effective concentration of PS20 in the anti-CD79b-vc-MMAE formulation. The bags were briefly agitated at 30°C for 30 minutes. This short agitation at 30°C covers the agitation conditions that diluted anti-CD79b-vc-MMAE might encounter during transport under ambient conditions in hospitals or other facilities. The study used IV infusion bags with concentrations of 0.001%, 0.002%, 0.003%, and 0.005% PS20, and agitated at 100 rpm at 30°C. The results are shown in Figure 6. Based on these results, the increase was less than 0.1% when the bag contained 0.005% PS20. Under these conditions, the increase in HMWS for anti-CD79b-vc-MMAE was directly related to the concentration of PS20 in the IV infusion bag. Based on the results of this study, in order to ensure the physical stability of the diluted anti-CD79b-vc-MMAE in IV infusion bags, the minimum content of PS20 in IV infusion bags should be between 0.003% and 0.005% to keep the aggregate level below the lower limit of quantitation.

[0421] Stir the IV bag at 2-8°C while cooling (do not pre-cool the bag):

[0422] The aim of this study was to support the transport of compound IV infusion bags under more realistic conditions. Typically, IV infusion bags are compounded by pharmacists and transported in vehicles, either passing through or not pre-cooled. Prior to this experiment, the bags were equilibrated to 30°C to simulate higher ambient temperatures that may exist around the world. This ensures the stability of the diluted DP even when transported in uncooled bags or containers. The study was conducted using 2.6 mg / mL of anti-CD79b-vc-MMAE in 100 mL IV infusion bags with four different PS20 concentrations: 0.001%, 0.002%, 0.003%, and 0.005%. The HMWS increase was less than 0.1% when the concentration in the IV infusion bag was ≤0.002%. Based on the results of this stirring model study, the minimum PS20 concentration in IV infusion bags is between 0.001% and 0.002% to keep aggregate levels below the lower limit of quantitation. The results are summarized in Figure 7.

[0423] in conclusion:

[0424] Antibody-drug conjugates (ADCs) are more sensitive to saline and high ionic strength buffers than conventional unconjugated antibodies (Adem, Y. et al., Bioconjugate Chemistry, 25 (2014) 656-664). Testing of anti-CD79b-vc-MMAE showed an increase in HMWS after 22 hours of static storage at 30°C when diluted to 0.3% saline.

[0425] Based on these two stirring models, anti-CD79b-vc-MMAE in IV infusion bags containing at least 0.002% PS20 was stably stored for 1 hour at 2-8°C under stirring conditions induced by short-term transport. To meet the stability requirements under stirring stress during handling, transport, and short-term storage, the final proposed anti-CD79b-vc-MMAE formulation contained a surfactant concentration of 0.12%, which, when used in combination with a 100 mL IV infusion bag, resulted in a content of 0.004% in the bag for the expected minimum patient weight (40 kg). This surfactant level demonstrated protective activity against anti-CD79b-vc-MMAE at doses ranging from 1.8 mg / kg to 2.4 mg / kg. It is noteworthy that, considering the following scientific literature, the level of surfactant (especially PS20) is considered unexpectedly high and counterintuitive, see: Kerwin BA. Polysorbates 20 and 80 used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways. Journal of Pharmaceutical Sciences. 2008;97(8):2924-2935. The amount of PS20 used in anti-CD79b-vc-MMAE drugs is also unexpectedly high compared to another immunoconjugate anti-CD22-vc-MMAE, and commercial formulations containing 0.05% PS20 for anti-CD22-vc-MMAE were determined to be suitable for dilution in IV infusion bags (data not shown).

[0426] Example 2: Reducing the risk of oxidation and linker hydrolysis of anti-CD79b immunoconjugates

[0427] As described in Example 1, determining the appropriate surfactant concentration for IV bag delivery may increase the problem of anti-CD79b-vc-MMAE oxidation. Higher polysorbate contents are known to be associated with higher peroxide contents due to the auto-oxidative degradation of polysorbates. See: Lam XM, Yang JY, Cleland JL. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. Journal of Pharmaceutical Sciences. 1997;86(11): 1250-1255; Donbrow M, Azaz E, Pillersdorf A. Autoxidation of polysorbates. Journal of Pharmaceutical Sciences. 1978;67(12): 1676-1681; Kerwin BA. Polysorbates 20 and 80 used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways. Journal of Pharmaceutical Sciences. 2008;97(8):2924-2935.

[0428] Furthermore, tryptophan oxidation is known to occur in the presence of PS20. See Lam X et al., Site-Specific Tryptophan Oxidation Induced by Autocatalytic Reaction of Polysorbate 20 in Protein Formulation, Pharm Res (2011) 28: 2543-2555. Notably, for the anti-CD79b antibody used in this disclosure, tryptophan is present in the variable heavy chain HVR 1: GYTFSSYWIE (SEQ ID NO:4). After formulation of anti-CD79b-vc-MMAE with AAPH, the oxidation tendency under AAPH stress conditions (e.g., storage at 40°C for up to 2 weeks, at 25°C for up to 6 months, or at 2-8°C for 6 months) was determined by mass spectrometry analysis of trypsin peptides. Stressed anti-CD79b-vc-MMAE was digested with trypsin, and the percentage of oxidation of the digested peptides was determined by LC-MS / MS (data not shown). Peptide mapping analysis revealed that 68% of HVR tryptophan was oxidized, resulting in approximately 58% loss of anti-CD79b-vc-MMAE potency (data not shown). Therefore, it is necessary to reduce the risk of oxidation.

[0429] Other factors to consider include how to control the succinimide hydrolysis of the immunoconjugate linker. The anti-CD79b-vc-MMAE immunoconjugate employs a maleimide-containing drug linker that couples to the cysteine ​​residues of the antibody to form a thiosuccinimide bond. The thiosuccinimide bond is known to undergo two competing reactions in patient plasma: (a) maleimide elimination, leading to undesirable drug loss from the immunoconjugate; and (b) thiosuccinimide ring hydrolysis, resulting in the inability to eliminate the succinic acid derivative. See Figure 8 and Lyon, R. et al., Self-Stabilizing ADCs: Conjugates Prepared with Maleimido Drug-Linkers that Catalyze their own Thiosuccinimide Ring Hydrolysis, Abstract No. 4333, American Association for Cancer Research (April 2013). This is an important critical quality attribute (CQA) because it can affect pharmacokinetics (PK) and safety. It has been previously demonstrated that initial liquid formulations used in early clinical trials in patients, such as those in histidine acetate buffer at pH 5.5, showed an increase in acidity of approximately 9% after 2 years of storage under recommended conditions (data not shown).

[0430] Because of the following three risks: increased PS20 concentration leading to increased oxidation risk; increased tryptophan oxidation leading to increased oxidation risk and resulting in loss of potency; and linker hydrolysis leading to loss of MMAE drug in the immunoconjugate and the generation of free drug in the patient's plasma, it is recommended to use lyophilized formulations to reduce these risks and thus obtain a longer and more stable shelf life.

[0431] A. Research on alternative buffer solutions:

[0432] To initiate the formulation development of the lyophilized product, alternative buffer solutions were investigated. The buffer solution used in the early clinical formulation was histidine acetate buffer. L-histidine is known to be oxidized through various mechanisms, the most common being photo-oxidation. See Mason, B. et al., Oxidation of Free L-histidine by tert-Butylhydroperoxide, *Pharm Res.* (2010) 27(3): 447-456. Furthermore, acetate may evaporate during lyophilization, leading to pH changes, which could destabilize the immunoconjugate product. Additionally, acetate is flammable and is considered a potential hazard to the lyophilization process. Therefore, alternative buffer solutions were evaluated. The three formulations tested are shown in Table 3.

[0433] Table 3: Buffer type screening and pH range assessment

[0434]

[0435] In freeze-thaw stress tests, the stability of the freeze-thawed anti-CD79b-vc-MMAE drug composition (see Table 3) in disposable PETG containers was evaluated. The protein formulation was aseptically filtered and filled into PETG containers. The containers were placed at -20°C for T=0 and 1 week. The containers were then frozen and thawed to room temperature for a total of five cycles. Only the T=0 and third freeze-thaw cycles were analyzed. The results are summarized in Table 4.

[0436] Table 4: Freeze-thaw stability against CD79b-vc-MMAE

[0437]

[0438] The results showed that no problems were observed in any of the three formulations tested. Therefore, histidine or succinate can be considered acceptable alternative buffer types for histidine-acetate.

[0439] Next, in the stability screening test, the effect of pH at 30°C on charge variant formation was investigated. Using the same three liquid formulations as those in Table 3, the samples were subjected to stress at 30°C for three different time points of 2, 4, and 8 weeks. Charge variant distribution was assessed using an iCE280 analyzer (ProteinSimple) equipped with a PrinCE microsyringe and a 100 µm × 5 cm fluorocarbon-coated capillary column (ProteinSimple). To remove the C-terminal lysine residues of the heavy chain, carboxypeptidase B (CpB) was added to each sample at a 1:1000 (w / w) enzyme-to-substrate ratio after the dilution step. Additionally, sialidase A was added to remove sialic acid. After the addition of CpB and sialidase A, the samples were incubated at 37°C for 10 minutes. The incubated liquid formulation sample was mixed with an amphoteric electrolyte solution consisting of 700 µL 1% methylcellulose, 1218 µL purified water, 8 µL pharmalyte 8-10.5, 55 µL pharmalyte 5-8, 15 µL pI-labeled 5.12, and 4 µL pI-labeled 7.05. The sample was focused using an 80 mM phosphoric acid anolyte and a 100 mM sodium hydroxide catholyte (both dissolved in 0.1% methylcellulose), with a potential of 1500 V introduced and held for 1 minute, followed by a potential of 3000 V introduced and held for 5 minutes. Images of the focused charge variants were obtained by passing 280 nm ultraviolet (UV) light through a capillary and into the lens of a charge-coupled device (CCD) digital camera. The results, shown in Figures 9 and 10, indicate that less acidic material is formed between pH 5.0 and 5.5, while the formation of alkaline material is slightly faster. Therefore, the composition of the buffer solution appears to have no measurable effect on charge stability.

[0440] B. Study on the effect of pH on stability:

[0441] In the stability screening, the same three liquid formulations as those in Table 3 were used to test the effect of pH on size variant formation at 30°C. These liquid formulations were subjected to stress at 30°C for three different time points: 2 weeks, 4 weeks, and 8 weeks. Size variant distribution was determined by size exclusion chromatography (SEC) using a mobile phase of 0.25 mM potassium chloride (pH 6.2) on an Agilent Technologies 1200 series HPLC (Santa Clara, California). The liquid formulation samples were then loaded onto a Tosoh Bioscience TSKgel G3000SWXL column (South San Francisco, California). The samples were eluted over 30 minutes at a flow rate of 0.5 mL / min, and the absorbance at 280 nm was monitored. The results are reported as relative peak areas of the total area under the curve, as shown in Figure 11. The stability screening results indicate that fewer high molecular weight substances (HMWS) were observed between pH 5.0 and 5.5 compared to pH 6.0. Similar amounts of low molecular weight substances (LMWS) were observed after 4 weeks of storage at 30°C. Slightly higher LMWS were observed at 30°C after 8 weeks. pH 5.3 was chosen because lowering the pH to 5.3 appeared to reduce the formation of charge and size variants.

[0442] Figure 12 shows the chromatograms obtained from testing two types of buffers: histidine / histidine-HCl at pH 5.5 and sodium succinate at three different pH values: 5.0, 5.5, and 6.0. At any pH tested (pH 5.0, 5.5, or 6.0), the histidine buffer at pH 5.5 showed slightly better physical stability than sodium succinate at 30°C. However, sodium succinate buffer was chosen because it exhibited acceptable stability in liquid and considering the potential oxidation risk associated with using histidine.

[0443] C. Testing lyophilized formulations:

[0444] The lyophilized formulation was tested to mitigate the aforementioned risks. The overall protocol for the lyophilization cycle is shown in Table 5.

[0445] Table 5: Lyophilization cycles for resistance to CD79b-vc-MMAE

[0446]

[0447] The cycle length is approximately 78 hours or approximately 3 days. Set the moisture content to between 0.5% and 0.7%.

[0448] A freeze-drying screening study was conducted, testing various concentrations of anti-CD79b-vc-MMAE, two different types of buffer solutions, different pH values, and two different surfactant levels, as shown in Table 6.

[0449] Table 6: Lyophilization Screening Study – Test Formulations

[0450]

[0451] In the screening studies, these lyophilized formulations were able to be stored stably at 30°C for up to 2 months.

[0452] To quantitatively analyze the monomers developed as stability indicators for the various test formulations (1) to (5), SEC analysis was performed when the various test formulations were reconstituted to 10 mg / mL or 20 mg / mL, as described above. Figure 13 summarizes this study, showing that the percentage of monomers in any of the test formulations remained unchanged after 2 months of heat stress at 30°C. As described above, the various lyophilized formulations (1) to (5) were also tested by icIEF to determine the percentage of the main peak after 2 months of heat stress at 30°C. Figure 14 summarizes this study and demonstrates that the percentage of the main peak remained unchanged after 2 months of heat stress at 30°C for any of the test formulations. The results of these two studies indicate that the tested lyophilized formulations have acceptable stability in the pH range of 5.0 to 6.0 using sodium succinate buffer.

[0453] D. Appearance of freeze-dried block products:

[0454] An important aspect of lyophilized formulations is the actual appearance of the lyophilized blocks, as it is one of the product quality attributes. For the anti-CD79b-vc-MMAE lyophilized formulation, the concentrations of the immunoconjugate and the stabilizer / anti-tensile agent were tested simultaneously to examine how to improve the appearance of the lyophilized blocks. Figures 15A to 15C show three lyophilized formulations with different protein-to-sucrose ratios tested. At all three sucrose contents tested, indentations appeared at the bottom of the lyophilized blocks, failing to achieve the desired appearance. However, under similar lyophilization conditions, the formulation with lower sucrose content exhibited smaller indentations, and lower sucrose content resulted in lower moisture content. Another lyophilized formulation was tested where the immunoconjugate concentration was increased to 20 mg / mL, while the sucrose concentration was reduced to 120 mM. Figures 16A and 16B compare the appearance of the two lyophilized blocks. The 20 mg / mL immunoconjugate combined with 120 mM sucrose exhibited the most stable and uniform appearance of the lyophilized blocks. During a 4-week heat stress stability study at 40°C, 25°C, and 5°C, no changes were observed in the structure, color, or appearance of the blocks, and the moisture content remained below 5%.

[0455] E. Stability of lyophilized formulations:

[0456] Lyophilized pharmaceutical formulations: The stability of 20 mg / mL anti-CD79b-vc-MMAE in 120 mM sucrose and 10 mM sodium succinate with 0.12% PS20 (pH 5.3) was tested by the formation of soluble aggregates or charge variants. Soluble aggregates were measured by SEC as described above. Figure 17 shows that for the lyophilized pharmaceutical formulation, the increase in soluble aggregates was less than 0.1% over 8 weeks of storage at 25°C and 2–8°C. At 40°C, an increase in dimers was observed weekly of approximately 0.1%, and this aggregation rate may depend on the moisture content. Charge variants were measured by icIEF as described above. Figure 18 shows that for the lyophilized pharmaceutical formulation, after 4 weeks of storage at 40°C, the main peak loss was approximately 5%, and the acidity increased by 3.7%. No significant changes were observed at 25°C and 2–8°C. Therefore, no changes were observed in the lyophilized drug under stress conditions of 25°C and 2-8°C.

[0457] Table 7 summarizes the final anti-CD79b-vc-MMAE formulations derived from the numerous studies described in Examples 1 and 2.

[0458] Table 7: Summary of the final anti-CD79b-vc-MMAE formulation

[0459]

[0460] As shown in Table 7 above, the final commercial formulation contains 20 mg / mL of the drug. When the solution is lyophilized, the increased protein-to-drug ratio improves the appearance of the bulk. 0.12% PS20 (i.e., 1.2 mg / mL) allows for the use of IV infusion bags and intravenous sets to protect the protein in the formulation from interfacial stress during administration. A pH of 5.3 minimizes the formation of acidic variants resulting from succinimide hydrolysis. The formulation shown in Table 7 is designed to deliver 140 mg of the drug per vial after reconstitution in 7.2 mL SWFI, supporting a clinical dose of 1.8 mg / kg.

[0461] Example 3: Evaluation of the stability of anti-CD79b-vc-MMAE active pharmaceutical ingredient and pharmaceutical formulation

[0462] The lyophilized formulations (see Table 7) were found to remain stable for at least 44 months at 2%–8°C with a residual moisture content ranging from 0.3% (w / w) to 3.2% (w / w), and at 25°C with a residual moisture content ranging from 0.3% (w / w) to 3.2% (w / w) for at least 7 months.

[0463] Next, the formulation parameters of the lyophilized drug were modified, and their practically meaningful impact on the stability of the lyophilized drug was assessed. Five formulation parameters were tested (and the range of parameter variations): (a) protein concentration (17–23 mg / mL), (b) succinate concentration (7–23 mM), (c) sucrose concentration (90–150 mM), (d) polysorbate 20 concentration (0.9–1.5 mg / mL), and (e) pH (4.95–5.65). Storage at 2–8°C for up to 9 months had no practically meaningful impact on the stability of the lyophilized drug. Based on the model-predicted degradation rate, storage at 2–8°C for at least 24 months was expected to have no practically meaningful impact on the stability of the lyophilized drug.

[0464] The reconstituted drug solution (i.e., 20 mg / mL polatuzumab vedotin, 120 mM sucrose, and 1.2 mg / mL polysorbate 20 in 10 mM succinate at pH 5.3) remained chemically stable after storage at 2–8°C for 72 hours or after storage at 30°C under ambient light exposure for 24 hours. No significant changes (e.g., statistically significant changes) were found in the affinity of polatuzumab vedotin for its target (CD79b) or in the bioactivity of polatuzumab vedotin.

[0465] Although this disclosure has been described in considerable detail for the purpose of clear understanding, such descriptions and examples should not be construed as limiting the scope of this disclosure. The entire contents of all patent and scientific literature disclosures cited herein are expressly incorporated by reference.

[0466] This disclosure relates to the following implementation plan.

[0467] 1. A pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant, wherein the concentration of the surfactant is at least 0.06% w / v, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0468]

[0469] in:

[0470] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and

[0471] The p-value is approximately 1 to approximately 8.

[0472] 2. The pharmaceutical composition according to claim 1, characterized in that the concentration of the anti-CD79b immunoconjugate is about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 30 mg / mL, or about 10 mg / mL to about 20 mg / mL.

[0473] 3. The pharmaceutical composition according to item 2, characterized in that the concentration of the anti-CD79b immunoconjugate is from about 10 mg / mL to about 20 mg / mL.

[0474] 4. The pharmaceutical composition according to item 2 or 3, characterized in that the concentration of the anti-CD79b immunoconjugate is 20 mg / mL.

[0475] 5. The pharmaceutical composition according to any one of claims 1-3, characterized in that the composition comprises the anti-CD79b immunoconjugate at a concentration of 10 mg / mL and the surfactant at a concentration of 0.06% w / v.

[0476] 6. The pharmaceutical composition according to any one of claims 1-4, characterized in that the composition comprises the anti-CD79b immunoconjugate at a concentration of 20 mg / mL and the surfactant at a concentration of 0.12% w / v.

[0477] 7. The pharmaceutical composition according to any one of claims 1-4, characterized in that the composition comprises the anti-CD79b immunoconjugate at a concentration of 20 mg / mL and the surfactant at a concentration of at least 0.12% w / v.

[0478] 8. The pharmaceutical composition according to any one of items 1-7, characterized in that the surfactant is selected from the group consisting of: polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188 (P188), N-octyl-β-D-glucopyranoside (OG), and combinations thereof.

[0479] 9. The pharmaceutical composition according to any one of claims 1-8, characterized in that the composition further comprises a buffer.

[0480] 10. The pharmaceutical composition according to item 9, characterized in that the buffer is a histidine buffer or a succinate buffer.

[0481] 11. The pharmaceutical composition according to item 10, characterized in that the buffer is a histidine buffer.

[0482] 12. The pharmaceutical composition according to item 10, characterized in that the buffer is a succinate buffer.

[0483] 13. The pharmaceutical composition according to item 12, characterized in that the succinate buffer is sodium succinate buffer.

[0484] 14. The pharmaceutical composition according to claim 13, characterized in that the concentration of the sodium succinate buffer is from about 10 mM to about 200 mM.

[0485] 15. The pharmaceutical composition according to claim 14, characterized in that the concentration of the sodium succinate buffer is 10 mM.

[0486] 16. The pharmaceutical composition according to any one of claims 1-15, characterized in that the composition has a pH of about 5.0 to about 6.0.

[0487] 17. The pharmaceutical composition according to claim 16, characterized in that the composition has a pH of 5.3.

[0488] 18. The pharmaceutical composition according to any one of items 1-17, characterized in that the composition further comprises sugar.

[0489] 19. The pharmaceutical composition according to claim 18, characterized in that the concentration of the sugar is from about 100 mM to about 260 mM.

[0490] 20. The pharmaceutical composition according to items 18-19, characterized in that the sugar is selected from the group consisting of: sucrose, mannitol, sorbitol, glycerol, dextran 40 and trehalose.

[0491] 21. The pharmaceutical composition according to item 20, characterized in that the sugar is sucrose.

[0492] 22. The pharmaceutical composition according to item 22, characterized in that the concentration of sucrose is 120 mM.

[0493] 23. The pharmaceutical composition according to any one of items 1-22, characterized in that the composition has been reconstituted from lyophilized blocks.

[0494] 24. A pharmaceutical composition prepared by lyophilization of a liquid composition comprising 20 mg / mL anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v polysorbate 20, and 120 mM sucrose, characterized in that the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0495]

[0496] in:

[0497] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and

[0498] The p-value is approximately 1 to approximately 8.

[0499] 25. The pharmaceutical composition according to any one of claims 1-24, characterized in that the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 8.

[0500] 26. The pharmaceutical composition according to any one of claims 1-25, characterized in that the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0501] 27. The pharmaceutical composition according to any one of items 1-26, characterized in that the p value is between about 2 and about 5.

[0502] 28. A pharmaceutical composition prepared by lyophilization of a liquid composition comprising 20 mg / mL of an anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v polysorbate 20, and 120 mM sucrose, wherein the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0503]

[0504] in:

[0505] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5.

[0506] 29. The pharmaceutical composition according to any one of items 1-28, characterized in that p is about 3.5.

[0507] 30. The pharmaceutical composition according to any one of items 24-29, characterized in that the composition remains stable for about 48 months when stored protected from light at 5°C ± 3°C.

[0508] 31. The pharmaceutical composition according to claim 30, characterized in that the stability of the composition is determined by size exclusion high-performance liquid chromatography (SE-HPLC).

[0509] 32. The pharmaceutical composition according to claim 31, characterized in that the composition has a main peak (peak area %) of at least 95.0 as determined by SE-HPLC.

[0510] 33. The pharmaceutical composition according to any one of items 28-32, characterized in that the stability of the composition is measured by imaging capillary isoelectric focusing (icIEF).

[0511] 34. The pharmaceutical composition according to claim 33, characterized in that the composition has a main peak of at least 58.0 (peak area %), an acidic region of up to 32.0 (peak area %), and a basic region of up to 12.0 (peak area %) as measured by icIEF.

[0512] 35. The pharmaceutical composition according to any one of items 24-34, characterized in that the pharmaceutical composition is a lyophilized block.

[0513] 36. The pharmaceutical composition according to any one of items 1-35, characterized in that the pharmaceutical composition is reconstituted with sterile water for injection (SWFI) to produce a reconstituted composition.

[0514] 37. The pharmaceutical composition according to any one of items 1-35, characterized in that the pharmaceutical composition is reconstituted in about 7.2 mL of SWFI to produce a reconstituted composition.

[0515] 38. The pharmaceutical composition according to item 36 or 37, characterized in that the reconstituted composition can be stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0516] 39. The pharmaceutical composition according to any one of claims 36-38, characterized in that the reconstituted composition can be stored stably at about 2°C to about 8°C for at least 24, 48 or 72 hours.

[0517] 40. The pharmaceutical composition according to any one of claims 36-39, characterized in that the reconstituted composition can be further diluted in an isotonic buffer solution in an intravenous (IV) infusion bag to produce a diluted composition.

[0518] 41. The pharmaceutical composition according to claim 40, characterized in that the final volume of the diluted composition in the IV infusion bag is about 100 mL.

[0519] 42. The pharmaceutical composition according to item 40 or 41, characterized in that the concentration of the immunoconjugate in the diluted composition is between about 0.72 mg / mL and about 2.7 mg / mL.

[0520] 43. A pharmaceutical composition prepared by a method comprising the following steps:

[0521] (a) Lyophilizing a liquid composition to produce a lyophilized composition, said liquid composition comprising 20 mg / mL anti-CD79b immunoconjugate in 10 mM sodium succinate buffer, 0.12% w / v polysorbate 20, and 120 mM sucrose, wherein said liquid composition has a pH of 5.3, and wherein said anti-CD79b immunoconjugate comprises the formula:

[0522]

[0523] in:

[0524] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5;

[0525] (b) Reconstitute the lyophilized composition with approximately 7.2 mL of sterile water for injection (SWFI) to produce a reconstituted composition; and

[0526] (c) The reconstituted composition is diluted in isotonic buffer solution in an intravenous (IV) infusion bag to produce the pharmaceutical composition.

[0527] The final volume of the drug composition in the IV infusion bag is approximately 100 mL, and

[0528] The final concentration of the immunoconjugate in the pharmaceutical composition is about 0.72 mg / mL or about 2.7 mg / mL.

[0529] 44. The pharmaceutical composition according to item 43, characterized in that p is about 3.5.

[0530] 45. The pharmaceutical composition according to any one of claims 38-44, characterized in that the isotonic buffer solution is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, or a 5% glucose solution.

[0531] 46. ​​A liquid composition comprising: a) a concentration of polatuzumabvedotin in the range of about 0.72-2.7 mg / mL; b) a concentration of sodium succinate in the range of about 0.36-1.35 mM; c) a concentration of sucrose in the range of about 0.51-16.24 mM; and d) a concentration of polysorbate 20 in the range of about 0.0432-0.162 mg / mL, wherein the pH of the liquid composition is between about 5 and about 5.7.

[0532] 47. A liquid composition comprising: a) about 0.72 mg / mL polatuzumab vedotin; b) about 0.36 mM sodium succinate; c) about 0.51 mM sucrose; d) about 0.0432 mg / mL polysorbate 20, wherein the pH of the liquid composition is between 5.1 and about 5.4.

[0533] 48. A liquid composition comprising: a) about 2.7 mg / mL polatuzumab vedotin; b) about 0.35 mM sodium succinate; c) about 16.24 mM sucrose; d) about 0.162 mg / mL polysorbate 20, wherein the pH of the liquid composition is between about 5.1 and about 5.4.

[0534] 49. The liquid composition according to any one of claims 46-48, characterized in that the volume of the liquid composition is between about 50 mL and about 100 mL.

[0535] 50. The liquid composition according to claim 49, characterized in that the volume of the liquid composition is 50 mL.

[0536] 51. The liquid composition according to claim 49, characterized in that the volume of the liquid composition is 100 mL.

[0537] 52. A pharmaceutical composition comprising an anti-CD79b immunoconjugate, a surfactant, a succinate buffer, and a sugar, wherein the pharmaceutical composition, when reconstituted in water, forms a liquid pharmaceutical composition, the liquid pharmaceutical composition comprising the anti-CD79b immunoconjugate at a concentration of about 10 mg / mL to about 20 mg / mL, the surfactant at a concentration of at least 0.06% w / v, the succinate buffer at a concentration of about 10 mM to about 200 mM, and the sugar at a concentration of about 100 mM to about 260 mM, wherein the liquid pharmaceutical composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0538]

[0539] in:

[0540] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and

[0541] The p-value is approximately 1 to approximately 8.

[0542] 53. The pharmaceutical composition according to claim 52, characterized in that the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 8.

[0543] 54. The pharmaceutical composition according to item 52 or 53, characterized in that the heavy chain of the anti-CD79b comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10.

[0544] 55. The pharmaceutical composition according to any one of items 24-34 or 52-54, characterized in that the pharmaceutical composition is prepared by lyophilization, and wherein the lyophilized pharmaceutical composition is stably stored at about 2°C to about 8°C for at least 6, 12, 18, 24, 30, 36, 42, 48, 54 or 60 months.

[0545] 56. The pharmaceutical composition according to claim 55, characterized in that the lyophilized pharmaceutical composition is a lyophilized block.

[0546] 57. The pharmaceutical composition according to any one of claims 52-56, characterized in that the pharmaceutical composition, after reconstitution, can be stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0547] 58. The pharmaceutical composition according to any one of claims 52-57, characterized in that the pharmaceutical composition, after reconstitution, can be stably stored at about 2°C to about 8°C for at least 24, 48, or 72 hours.

[0548] 59. The pharmaceutical composition according to any one of claims 52-58, characterized in that the pharmaceutical composition is diluted in an isotonic buffer in an IV infusion bag after reconstitution, wherein the concentration of the surfactant in the IV infusion bag after dilution is at least 0.003% w / v.

[0549] 60. The pharmaceutical composition according to claim 59, characterized in that the concentration of the surfactant diluted in the IV infusion bag is at least 0.004% w / v.

[0550] 61. The pharmaceutical composition according to any one of claims 52-60, characterized in that the surfactant is polysorbate 20.

[0551] 62. The pharmaceutical composition according to any one of items 52-61, characterized in that the sugar is sucrose.

[0552] 63. The pharmaceutical composition according to any one of items 52-58 and 61-62, characterized in that the concentration of sucrose is 120 mM.

[0553] 64. The pharmaceutical composition according to any one of claims 52-63, characterized in that the succinate buffer is sodium succinate buffer.

[0554] 65. The pharmaceutical composition according to item 64, characterized in that, after reconstitution but before dilution, the concentration of the sodium succinate buffer is 10 mM.

[0555] 66. The pharmaceutical composition according to any one of items 52-58 and 61-65, characterized in that the composition remains stable at 5°C ± 3°C for about 7 days.

[0556] 67. The pharmaceutical composition according to claim 66, characterized in that the stability of the composition is determined by size exclusion high-performance liquid chromatography (SE-HPLC).

[0557] 68. The pharmaceutical composition according to claim 67, characterized in that the composition has a main peak (peak area %) of at least 95.0 as determined by SE-HPLC.

[0558] 69. The pharmaceutical composition according to any one of claims 52-68, characterized in that the stability of the composition is measured by imaging capillary isoelectric focusing (icIEF).

[0559] 70. The pharmaceutical composition according to claim 69, characterized in that the composition has a main peak of at least 58.0 (peak area %), an acidic region of up to 32.0 (peak area %), and an alkaline region of up to 12.0 (peak area %) as measured by icIEF.

[0560] 71. A glass vial comprising a pharmaceutical composition according to any one of claims 52-58 and 61-70.

[0561] 72. A liquid composition comprising an anti-CD79b immunoconjugate, a surfactant, a succinate buffer, and a sugar, wherein the concentration of the anti-CD79b immunoconjugate is from about 10 mg / mL to about 20 mg / mL, the concentration of the surfactant is at least 0.06% w / v, the concentration of the succinate buffer is from about 10 mM to about 200 mM, and the concentration of the sugar is from about 100 mM to about 260 mM, wherein the liquid composition has a pH of 5.3, and wherein the anti-CD79b immunoconjugate comprises the formula:

[0562]

[0563] in:

[0564] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and

[0565] The p-value is approximately 1 to approximately 8.

[0566] 73. The liquid composition according to claim 72, characterized in that the anti-CD79b antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 8.

[0567] 74. The liquid composition according to item 72 or 73, characterized in that the heavy chain of the anti-CD79b comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10.

[0568] 75. The liquid composition according to any one of items 72-74, characterized in that the surfactant is polysorbate 20.

[0569] 76. The liquid composition according to any one of items 72-75, characterized in that the sugar is sucrose.

[0570] 77. The liquid composition according to claim 76, characterized in that the concentration of the sucrose is about 120 mM.

[0571] 78. The liquid composition according to any one of items 72-77, characterized in that the succinate buffer is a sodium succinate buffer.

[0572] 79. The liquid composition according to claim 78, characterized in that the concentration of the sodium succinate buffer is 10 mM.

[0573] 80. The liquid composition according to any one of claims 72-79, characterized in that the composition is diluted in an isotonic buffer solution.

[0574] 81. The liquid composition according to claim 80, characterized in that the isotonic buffer is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, or a 5% glucose solution.

[0575] 82. The liquid composition according to item 80 or 81, characterized in that the composition diluted in isotonic buffer is in an IV infusion bag.

[0576] 83. The liquid composition according to any one of items 72-79, characterized in that the composition maintains stability for about 72 hours at 5°C ± 3°C.

[0577] 84. The liquid composition according to any one of items 72-79, characterized in that the composition remains stable at 25°C for about 24 hours.

[0578] 85. The liquid composition according to any one of claims 72-79, characterized in that the liquid composition can be stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0579] 86. The liquid composition according to any one of claims 72-79, characterized in that the liquid composition can be stably stored at about 2°C to about 8°C for at least 24, 48 or 72 hours.

[0580] 87. A lyophilized pharmaceutical composition comprising about 150 mg of an anti-CD79b immunoconjugate, about 9.0 mg of polysorbate 20, about 8.88 mg of succinic acid, about 4.08 mg of sodium hydroxide, and about 309 mg of sucrose, wherein the anti-CD79b immunoconjugate comprises the formula:

[0581]

[0582] in:

[0583] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and

[0584] The p-value is approximately 2 to approximately 5.

[0585] 88. A lyophilized pharmaceutical composition comprising about 150 mg of polatuzumab vedotin, about 9.0 mg of polysorbate 20, about 8.88 mg of succinic acid, about 4.08 mg of sodium hydroxide and about 309 mg of sucrose.

[0586] 89. A lyophilized pharmaceutical composition comprising about 140 mg of an anti-CD79b immunoconjugate, about 8.4 mg of polysorbate 20, about 8.27 mg of succinic acid, about 3.80 mg of sodium hydroxide, and about 288 mg of sucrose, wherein the anti-CD79b immunoconjugate comprises the formula:

[0587]

[0588] in:

[0589] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and

[0590] The p-value is approximately 2 to approximately 5.

[0591] 90. A lyophilized pharmaceutical composition comprising about 140 mg of polatuzumab vedotin, about 8.4 mg of polysorbate 20, about 8.27 mg of succinic acid, about 3.80 mg of sodium hydroxide and about 288 mg of sucrose.

[0592] 91. A lyophilized pharmaceutical composition comprising about 30 mg of an anti-CD79b immunoconjugate, about 1.8 mg of polysorbate 20, about 1.77 mg of succinic acid, about 0.816 mg of sodium hydroxide, and about 61.8 mg of sucrose, wherein the anti-CD79b immunoconjugate comprises the formula:

[0593]

[0594] in:

[0595] Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the heavy chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 9, and wherein the light chain of the anti-CD79b antibody comprises the amino acid sequence shown in SEQ ID NO: 10; Val is valine; Cit is citrulline; and the p value is from about 2 to about 5.

[0596] 92. A lyophilized pharmaceutical composition comprising about 30 mg of polatuzumab vedotin, about 1.8 mg of polysorbate 20, about 1.77 mg of succinic acid, about 0.816 mg of sodium hydroxide and about 61.8 mg of sucrose.

[0597] 93. The freeze-dried composition according to any one of items 87, 89 or 91, characterized in that p is about 3.5.

[0598] 94. A lyophilized pharmaceutical composition comprising about 140-150 mg of polatuzumab vedotin, about 8.4-9.0 mg of polysorbate 20, about 8.27-8.88 mg of succinic acid, about 3.80-4.08 mg of sodium hydroxide and about 288-309 mg of sucrose.

[0599] 95. The lyophilized pharmaceutical composition according to any one of claims 87-94, characterized in that the lyophilized pharmaceutical composition can be stably stored at about 2°C to about 8°C for at least 6, 12, 18, 24, 30, 36, 42, 48, 54 or 60 months.

[0600] 96. The lyophilized pharmaceutical composition according to any one of claims 87-95, characterized in that the lyophilized pharmaceutical composition is a lyophilized block.

[0601] 97. A liquid pharmaceutical composition comprising: a) 5-60 mg / mL polatuzumab vedotin; b) 10-200 mM sodium succinate; c) 100-260 mM sucrose; d) 0.06-0.12% w / v polysorbate 20, wherein the pH of the liquid composition is between 5 and 6.

[0602] 98. The liquid composition according to claim 97, comprising: a) 10-55 mg / mL polatuzumabvedotin; b) 10-100 mM sodium succinate; c) 150-260 mM sucrose; d) 0.08-0.12% w / v polysorbate 20, characterized in that the pH of the liquid composition is between 5.1 and 5.6.

[0603] 99. The liquid composition according to claim 97, comprising: a) 15-40 mg / mL polatuzumabvedotin; b) 10-50 mM sodium succinate; c) 200-260 mM sucrose; d) 0.1-0.12% w / v polysorbate 20, characterized in that the pH of the liquid composition is between 5.2 and 5.4.

[0604] 100. A liquid pharmaceutical composition comprising: a) 20 mg / mL polatuzumab vedotin; b) 10 mM sodium succinate; c) 120 mM sucrose; d) 0.12% w / v polysorbate 20, wherein the pH of the liquid composition is about 5.3.

[0605] 101. The liquid composition according to any one of claims 97-100, characterized in that the liquid composition can be stably stored at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0606] 101. The liquid composition according to any one of claims 97-101, characterized in that the liquid composition can be stably stored at about 2°C to about 8°C for at least 24, 48 or 72 hours.

[0607] 102. A method of treating a patient with a proliferative disorder, the method comprising administering to the patient any of the preceding pharmaceutical compositions or liquid compositions.

[0608] 103. The method according to item 102, wherein the proliferative disease is cancer.

[0609] 104. The method according to item 103, wherein the cancer is a B-cell proliferative disorder.

[0610] 105. The method according to claim 104, characterized in that the B-cell proliferative disorders are selected from the group consisting of: lymphoma, myeloma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), aggressive NHL, indolent lymphoma, follicular lymphoma (FL), relapsed aggressive NHL, relapsed indolent NHL, relapsed NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma.

[0611] 106. The method according to claim 105, wherein the B-cell proliferative disorder is non-Hodgkin lymphoma (NHL).

[0612] 107. The method according to claim 105, wherein the B-cell proliferative disorder is diffuse large B-cell lymphoma (DLBCL).

[0613] 108. The method according to claim 105, wherein the B-cell proliferative disorder is relapsed NHL or refractory NHL.

[0614] 109. The method according to claim 105, wherein the B-cell proliferative disorder is follicular lymphoma (FL).

Claims

1. A pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant, wherein, The concentration of the surfactant is at least 0.06% w / v, and the anti-CD79b immunoconjugate comprises the formula: in: Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody comprises a heavy chain and a light chain, wherein the light chain comprises: (a) the HVR-L1 sequence shown in KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) the HVR-L2 sequence shown in AASNLES (SEQ ID NO: 2); and (c) the HVR-L3 sequence shown in QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain comprises: (a) the HVR-H1 sequence shown in GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence shown in GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence shown in TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and The p-value is approximately 1 to approximately 8.

2. The pharmaceutical composition according to claim 1, characterized in that, The concentration of the anti-CD79b immunoconjugate is about 5 mg / mL to about 60 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 30 mg / mL, or about 10 mg / mL to about 20 mg / mL.

3. The pharmaceutical composition according to claim 2, characterized in that, The concentration of the anti-CD79b immunoconjugate is from about 10 mg / mL to about 20 mg / mL.

4. The pharmaceutical composition according to claim 2 or 3, characterized in that, The concentration of the anti-CD79b immunoconjugate was 20 mg / mL.

5. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The composition comprises the anti-CD79b immunoconjugate at a concentration of 10 mg / mL and the surfactant at a concentration of 0.06% w / v.

6. The pharmaceutical composition according to any one of claims 1-4, characterized in that, The composition comprises the anti-CD79b immunoconjugate at a concentration of 20 mg / mL and the surfactant at a concentration of 0.12% w / v.

7. The pharmaceutical composition according to any one of claims 1-4, characterized in that, The composition comprises the anti-CD79b immunoconjugate at a concentration of 20 mg / mL and the surfactant at a concentration of at least 0.12% w / v.

8. The pharmaceutical composition according to any one of claims 1-7, characterized in that, The surfactant is selected from the group consisting of: polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188 (P188), N-octyl-β-D-glucopyranoside (OG), and combinations thereof.

9. The pharmaceutical composition according to any one of claims 1-8, characterized in that, The composition further comprises a buffer.

10. The pharmaceutical composition according to claim 9, characterized in that, The buffer is a histidine buffer or a succinate buffer.

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