Stable Anti-CD79b immunoconjugate formulations

Abstract

To provide stable pharmaceutical compositions comprising an anti-CD79b immunoconjugate and a surfactant.SOLUTION: Disclosed is a liquid composition comprising an anti-CD79b immunoconjugate, a buffer, a saccharide and a surfactant, where the surfactant is a nonionic surfactant at a concentration of at least 1.2 mg / ml, the anti-CD79b immunoconjugate comprises a formula in which Ab is an anti-CD79b antibody comprising a heavy chain and light chain where the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN, (b) an HVR-L2 sequence of AASNLES, and (c) an HVR-L3 sequence of QQSNEDPLT; and the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE, (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG, and (c) an HVR-H3 sequence of TRRVPIRLDY, and p is a value from about 1 to about 8.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 657,185, filed April 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 146392044340SEQLIST.TXT, Recorded: April 9, 2019, Size: 12KB).

[0003] The present disclosure is directed to stable pharmaceutical compositions comprising an anti-CD79b immunoconjugate and a surfactant. The present disclosure also provides methods for using such compositions for the treatment of cancer. [Background technology]

[0004] Intravenous (IV) administration using an infusion bag is the most common delivery route for biologics in the commercial environment. To support the delivery and compatibility of biologics, such as immunoconjugates, via IV administration, it is necessary to design suitable therapeutic formulations that maintain stability after dilution into the infusion bag, during transportation, and throughout the entire course of administration (Bardin, C. et al. Annales pharmaceutiques francaises 69 (2011) 221-231).

[0005] One challenge of administering biologics, especially using IV infusion bags, is that the construction materials and infusion solutions can present an environment that destabilizes therapeutic proteins. In addition, proteins can also encounter interfacial stresses within the IV administration bag. Protein adsorption at solid-liquid and air-water interfaces can cause protein denaturation at the surface, resulting in protein aggregation (Shieh, I., et al. Mol Pharm. 12(2015):3184-93; Sreedhara, A., et al. Pharm. Sci. 101(2012):21-30). To further complicate IV administration, agitation of the infusion bag causes continuous renewal of the air-liquid interface, resulting in repeated damage to the protein over time. Aggregation caused by either solution conditions or interfacial stress that destabilize the IV administration bag can significantly negatively impact the product quality, efficacy, and immunogenicity of biologics.

[0006] The adverse effects of IV administration on biologics can be partially mitigated through the use of surfactants in formulation development. Non-ionic surfactants such as polysorbate-20 (PS20) or polysorbate-80 (PS80) are commonly used in protein formulations to protect and stabilize molecules against air-water interface stress (Kerwin, BA. J. Pharm. Sci. 97 (2008) 2924-2935). Surfactants can protect drug products (DPs) against surface-induced damage by competing with proteins at both air-water and solid-water interfaces (Kerwin, BA. J. Pharm. Sci. 97 (2008) 2924-2935). In addition, surfactants also reduce the surface tension of the system (Cleland, JL., et al. Critical reviews in therapeutic drug carrier systems 10 (1993) 307-377). However, a significant drawback of using non-ionic surfactants at concentrations suitable for IV administration bag delivery is that prolonged exposure of biologics to the surfactants can result in oxidation of certain amino acid residues, thereby reducing therapeutic efficacy. Lam X, et al., Pharm Res. (2011) 28: 2543-2555.

[0007] Importantly, biologics must also have a long, stable shelf life under storage conditions without compromising the structure and activity of the therapeutic protein prior to IV administration. For example, liquid formulations of antibody-drug conjugates (ADCs) that use linkers may be susceptible to acid-catalyzed hydrolysis of the linker during storage. Such instability can cause premature release of the drug compound upon IV administration to patients, negatively impacting the pharmacokinetics and safety of the biologic.

[0008] Thus, there exists a need for the development of stable pharmaceutical compositions that are stable for IV administration and have an extended shelf life under storage conditions. The present disclosure fulfills this need and provides other related benefits. Summary of the Invention

[0009] In one aspect, the disclosure provides a pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant, wherein the surfactant is at a concentration of at least 0.06% weight / volume (i.e., 0.6 mg / ml), and the anti-CD79b immunoconjugate has the formula: TIFF2025032097000001.tif25170, wherein The pharmaceutical composition is provided wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p is a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5).

[0010] In some embodiments, the anti-CD79b immune complex is at a concentration of 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.

[0011] In some embodiments, the anti-CD79b immune complex is at a concentration of about 10 mg / ml to about 20 mg / ml. In one embodiment, the anti-CD79b immune complex is at a concentration of about 10 mg / ml. In another embodiment, the anti-CD79b immune complex is at a concentration of about 20 mg / ml.

[0012] In some embodiments, the anti-CD79b immunoconjugate is at a concentration of 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.

[0013] In some embodiments, the anti-CD79b immune complex is at a concentration of 10 mg / ml to 20 mg / ml. In one embodiment, the anti-CD79b immune complex is at a concentration of 10 mg / ml. In another embodiment, the anti-CD79b immune complex is at a concentration of 20 mg / ml.

[0014] In some embodiments, the surfactant is at a concentration of about 0.06% by weight (i.e., 0.6 mg / ml) to about 0.12% by weight (i.e., 1.2 mg / ml). In some embodiments, the surfactant is at a concentration of at least about 0.06% by weight (i.e., 0.6 mg / ml). In another embodiment, the surfactant is at a concentration of at least about 0.12% by weight (i.e., 1.2 mg / ml). In some embodiments, the surfactant is at a concentration of at least 0.06% by weight (i.e., 0.12% by weight). In some embodiments, the surfactant is at a concentration of at least 0.12% by weight (i.e., 0.06% by weight). In another embodiment, the surfactant is at a concentration of 0.12% by weight.

[0015] In some embodiments, the anti-CD79b immune complex is at a concentration of about 10 mg / ml and the surfactant is at a concentration of about 0.06% by weight or volume (i.e., 0.6 mg / ml). In some embodiments, the anti-CD79b immune complex is at a concentration of about 20 mg / ml and the surfactant is at a concentration of at least about 0.12% by weight or volume (i.e., 1.2 mg / ml). In some embodiments, the anti-CD79b immune complex is at a concentration of about 20 mg / ml and the surfactant is at a concentration of about 0.12% by weight or volume (i.e., 1.2 mg / ml).

[0016] In some embodiments, the anti-CD79b immune complex is at a concentration of 10 mg / ml and the surfactant is at a concentration of 0.06% (i.e., 0.6 mg / ml). In some embodiments, the anti-CD79b immune complex is at a concentration of 20 mg / ml and the surfactant is at a concentration of at least 0.12% (w / v). In some embodiments, the anti-CD79b immune complex is at a concentration of 20 mg / ml and the surfactant is at a concentration of 0.12% (w / v).

[0017] In some embodiments according to (or as applied to) any of the above embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In some embodiments, the liquid pharmaceutical composition is stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at 30° C., or for at least about any of 24, 48, or 72 hours when stored at 2° C. to 8° C.

[0018] In certain embodiments of the present disclosure, the surfactant herein is non-ionic. In an 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 a specific embodiment, the surfactant is PS20. In yet another specific embodiment, the surfactant is PS80.

[0019] In some embodiments of the present disclosure, the composition further comprises a buffer. In certain embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a succinate buffer. In one particular embodiment, the succinate buffer is a sodium succinate buffer. In some embodiments, the sodium succinate buffer is at a concentration of about 10 mM to about 200 mM. In some embodiments, the sodium succinate buffer is at a concentration of 10 mM to 200 mM. In one embodiment, the sodium succinate buffer is at a concentration of about 10 mM. In another embodiment, the sodium succinate buffer is at a concentration of 10 mM.

[0020] In some embodiments, the composition of the present disclosure has a pH of about 5.0 to about 6.0. In certain embodiments, the buffer 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 composition of the present disclosure has a pH of about 5.3. In some embodiments, the composition of the present disclosure has a pH of 5.0 to 6.0. In certain embodiments, 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 embodiment, the composition of the present disclosure has a pH of 5.3.

[0021] In certain embodiments of the present disclosure, the composition further comprises a sugar. In some embodiments, the sugar is at a concentration of about 100 mM to about 260 mM. In some embodiments, the sugar is at a concentration of 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.

[0022] In one particular embodiment, the sugar is sucrose. In one embodiment of the present disclosure, the sucrose is at a concentration of about 120 mM. In another embodiment, the sucrose is at a concentration of 120 mM.

[0023] In some embodiments, the compositions of the present disclosure are lyophilized (such as a lyophilized cake). In some embodiments, the lyophilized composition is contained within a vial, for example, a 20 ml glass vial.

[0024] In one aspect, the disclosure provides a pharmaceutical composition produced by lyophilization of a liquid formulation comprising 20 mg / ml anti-CD79b immunoconjugate, 0.12% weight / volume polysorbate 20, and 120 mM sucrose in 10 mM sodium succinate buffer, wherein the liquid formulation has a pH of 5.3, and the anti-CD79b immunoconjugate has the formula: TIFF2025032097000002.tif24170, wherein The pharmaceutical composition is provided wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p is a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5).

[0025] In some embodiments of the disclosure, an anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 9 and the light chain comprises the amino acid sequence of SEQ ID NO: 10.

[0026] 1. A pharmaceutical composition produced by lyophilization of a liquid formulation comprising 20 mg / ml anti-CD79b immunoconjugate, 0.12% by weight / volume polysorbate 20, and 120 mM sucrose in 10 mM sodium succinate buffer, wherein the liquid formulation has a pH of 5.3, and the anti-CD79b immunoconjugate has the formula: Also provided is a pharmaceutical composition comprising the formula: TIFF2025032097000003.tif25170, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO:9, the light chain comprises the amino acid sequence of SEQ ID NO:10, Val is valine, Cit is citrulline, and p is a value of about 2 to about 5, such as about 3.5. In some embodiments, the pharmaceutical composition is a lyophilized cake.

[0027] In certain embodiments, the pharmaceutical compositions of the present disclosure have a stability of about 60 months when protected from light at 5° C.±3° C. In certain embodiments, the pharmaceutical compositions of the present disclosure have a stability of about 48 months when protected from light at 5° C.±3° C.

[0028] In some embodiments, the stability of the pharmaceutical composition of the present disclosure is measured by size exclusion chromatography high performance (SE-HPLC). In one embodiment, the composition has a main peak area % of at least 95.0 as measured by SE-HPLC.

[0029] In some embodiments, the stability of the pharmaceutical composition of the present disclosure is measured by imaging capillary isoelectric focusing (icIEF). In one embodiment, the composition has a main peak (area%) of at least 58.0, an acidic area (area%) of at most 32.0, and a basic area (area%) of at most 12.0, as measured by icIEF.

[0030] In certain embodiments, the pharmaceutical composition of the present disclosure is 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 stable for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at about 30° C. In some embodiments, the reconstituted composition is stable for at least 24, 48, or 72 hours when stored at about 2° C. to about 8° C. In some embodiments, the reconstituted composition is further diluted in an isotonic buffer in an intravenous (IV) administration bag. In some embodiments, the final volume of the diluted composition in the IV administration bag is about 50 ml to about 100 ml. In some embodiments, the concentration of the immune complex in the IV administration bag is about 0.72 mg to about 2.7 mg.

[0031] 1. A pharmaceutical composition produced by lyophilization of a liquid formulation comprising 20 mg / ml anti-CD79b immunoconjugate, 0.12% by weight / volume polysorbate 20, and 120 mM sucrose in 10 mM sodium succinate buffer, wherein the liquid formulation has a pH of 5.3, and the anti-CD79b immunoconjugate has the formula: Also provided is a pharmaceutical composition comprising the formula: TIFF2025032097000004.tif25170, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO:9, the light chain comprises the amino acid sequence of SEQ ID NO:10, Val is valine, Cit is citrulline, and p is a value of about 2 to about 5, such as about 3.5.

[0032] A liquid composition (e.g., for intravenous administration) is provided that comprises a) about 0.72 to about 2.7 mg / ml of polatuzumab vedotin, b) about 0.36 to about 01.35 mM of sodium succinate, c) about 0.51 to about 16.24 mM of sucrose, and d) about 0.0432 to about 0.162 mg / ml of polysorbate 20, and the pH of the liquid composition is about 5 to about 5.7. A liquid composition (e.g., for intravenous administration) is also provided that comprises a) about 0.72 mg / ml of polatuzumab vedotin, b) about 0.36 mM of sodium succinate, c) about 0.51 mM of sucrose, and d) about 0.0432 mg / ml of polysorbate 20, and the pH of the liquid composition is 5.1 to about 5.4. Also provided is a liquid composition (e.g., for intravenous administration) comprising a) about 2.7 mg / ml polatuzumab vedotin, b) about 0.1.35 mM sodium succinate, c) about 16.24 mM sucrose, and d) about 0.162 mg / ml polysorbate 20, wherein the pH of the liquid composition is about 5.1 to about 5.4. In some embodiments, the volume of the liquid composition is about 50 ml to 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) administration bag. In some embodiments, the surface of the IV administration bag in contact with the liquid composition is made of polyvinyl chloride (PVC), polyolefin (PO), polyethylene (PE), or polypropylene (PE).

[0033] In one aspect, the disclosure provides a pharmaceutical composition comprising an anti-CD79b immune conjugate, a surfactant, a succinate buffer, and a sugar, wherein the pharmaceutical composition, when reconstituted in water, forms a liquid formulation comprising an anti-CD79b immune conjugate at a concentration of about 10 mg / ml to about 20 mg / ml, a surfactant at a concentration of at least 0.06% weight by volume (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 the anti-CD79b immune conjugate is represented by the formula: TIFF2025032097000005.tif25170, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprising a heavy chain and a light chain, the light chain comprising (a) a HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) a HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) a HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), and the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), wherein Val is valine, Cit is citrulline, and p has a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5). In some embodiments, the anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10.

[0034] In some embodiments, the pharmaceutical composition is reconstituted in SWFI and then diluted into a buffer solution in an IV administration bag. In certain embodiments, the pharmaceutical composition is reconstituted in SWFI and then diluted into an isotonic buffer solution in an IV administration bag. In one embodiment, the surfactant concentration upon dilution into an IV administration bag is at least 0.003% by weight / volume. In one embodiment, the surfactant concentration upon dilution into an IV administration bag is at least 0.004% by weight / volume. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one particular embodiment, the sucrose is at a concentration of 120 mM. In some embodiments, the succinate buffer is a sodium succinate buffer. In one particular embodiment, the sodium succinate buffer is at a concentration of 10 mM. In some embodiments, the pharmaceutical composition is stable for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours upon storage at about 30° C. after reconstitution. In some embodiments, the pharmaceutical composition is stable for at least 24, 48, or 72 hours upon storage at about 2°C to about 8°C after reconstitution.

[0035] In some embodiments, the pharmaceutical composition has a stability of up to about 1 day, up to about 2 days, or up to about 3 days at 30° C. when reconstituted in water. In some embodiments, the pharmaceutical composition has a stability of up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, or up to about 7 days at 5° C.±3° C. when reconstituted in water. In certain embodiments, the stability of the composition is measured by size-exclusion high performance liquid chromatography (SE-HPLC). In one particular embodiment, the composition has at least 95.0 main peak (area %) as measured by SE-HPLC.

[0036] In certain embodiments, the stability of the composition is measured by imaging capillary isoelectric focusing (icIEF). In a particular embodiment, the composition has a main peak (area%) of at least 58.0, an acidic area (area%) of at most 32.0, and a basic area (area%) of at most 12.0, as measured by icIEF.

[0037] In some embodiments, the pharmaceutical compositions disclosed herein are contained within a glass vial (eg, a 20 ml glass vial).

[0038] In one aspect, the disclosure provides a liquid composition comprising an anti-CD79b immune complex, a surfactant, a succinate buffer, and a sugar, wherein the anti-CD79b immune complex is at a concentration of about 10 mg / ml to about 20 mg / ml, the surfactant is at a concentration of at least 0.06% weight / volume (i.e., 0.6 mg / ml), the succinate buffer is at a concentration of about 10 mM to about 200 mM, and the sugar is at a concentration of about 100 mM to about 260 mM, the liquid composition has a pH of 5.3, and the anti-CD79b immune complex is represented by the formula: TIFF2025032097000006.tif25170, wherein The pharmaceutical composition is provided wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p is a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5). In some embodiments, the anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10.

[0039] In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one particular embodiment, the sucrose is at a concentration of 120 mM. In some embodiments, the succinate buffer is a sodium succinate buffer. In one particular embodiment, the sodium succinate buffer is at a concentration of 10 mM.

[0040] In some embodiments, the liquid composition of the present disclosure is diluted in an isotonic buffer solution.In an exemplary embodiment, the liquid composition of the present disclosure is dissolved in an isotonic buffer solution in an IV administration bag.In some embodiments, the isotonic buffer solution in which the liquid composition is diluted is 0.9% sodium chloride solution, 0.45% sodium chloride solution, or 5% dextrose solution.

[0041] In some embodiments, the liquid compositions provided herein that are diluted in a 0.9% sodium chloride solution are stable (according to any one or more criteria described elsewhere herein) at 2°C to 8°C for up to about 24 hours after dilution. In some embodiments, the liquid compositions provided herein that are diluted in a 0.9% sodium chloride solution are stable (according to any one or more criteria described elsewhere herein) at 9°C to 25°C for up to about 4 hours after dilution. In some embodiments, the liquid compositions provided herein that are diluted in a 0.45% sodium chloride solution are stable (according to any one or more criteria described elsewhere herein) at 2°C to 8°C for up to about 24 hours after dilution. In some embodiments, the liquid compositions provided herein that are diluted in a 0.45% sodium chloride solution are stable (according to any one or more criteria described elsewhere herein) at 9°C to 25°C for up to about 4 hours after dilution. In some embodiments, the liquid compositions provided herein that are diluted in a 5% dextrose solution are stable (according to any one or more criteria described elsewhere herein) at 2°C to 8°C for up to about 48 hours after dilution. In some embodiments, the liquid compositions provided herein that are diluted in a 5% dextrose solution are stable (according to any one or more criteria described elsewhere herein) for up to about 8 hours at 9° C. to 25° C. after dilution.

[0042] In some embodiments, the liquid composition diluted in an isotonic buffer is present in an IV administration bag. In some embodiments, the surface of the IV administration bag that contacts the composition diluted in an isotonic buffer is composed of polyvinyl chloride (PVC), polyolefin (PO), polyethylene (PE), or polypropylene (PE).

[0043] In some embodiments, the liquid composition of the present disclosure diluted in an isotonic buffer in an IV administration bag has a stability of up to about 6 to about 8 hours at 30° C. In some embodiments, the liquid composition of the present disclosure dissolved in an isotonic buffer in an IV administration bag has a stability of up to about 24 hours at 25° C. In some embodiments, the liquid composition of the present disclosure dissolved in an isotonic buffer in an IV administration bag has a stability of up to about 72 hours at 5° C.±3° C. In certain embodiments, the isotonic buffer is normal saline. In some embodiments, the liquid composition is stable for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at about 30° C. In some embodiments, the liquid composition is stable for at least 24, 48, or 72 hours when stored at about 2° C. to about 8° C.

[0044] 1. 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 has the formula: Also provided is a lyophilized pharmaceutical composition comprising: TIFF2025032097000007.tif25170, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 9, the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, and p is a value of about 2 to about 5, such as about 3.5. In some embodiments, the lyophilized composition (such as a cake) is contained within a vial, for example a 20 ml glass vial.

[0045] Further provided is a lyophilized pharmaceutical composition comprising about 150 mg polatuzumab vedotin, about 9.0 mg polysorbate 20, about 8.88 mg succinic acid, about 4.08 mg sodium hydroxide, and about 309 mg sucrose. In some embodiments, the lyophilized composition (such as a cake) is contained within a vial, for example, a 20 ml glass vial.

[0046] 1. 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 has the formula: Also provided is a lyophilized pharmaceutical composition comprising: TIFF2025032097000008.tif25170, wherein b is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 9, the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, and p is a value of about 2 to about 5, such as about 3.5. In some embodiments, the lyophilized composition (such as a cake) is contained within a vial, for example a 20 ml glass vial.

[0047] Further provided is a lyophilized pharmaceutical composition comprising about 140 mg polatuzumab vedotin, about 8.4 mg polysorbate 20, about 8.27 mg succinic acid, about 3.80 mg sodium hydroxide, and about 288 mg sucrose. In some embodiments, the lyophilized composition (such as a cake) is contained within a vial, for example, a 20 ml glass vial.

[0048] 1. 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 has the formula: Also provided is a lyophilized pharmaceutical composition comprising the formula: TIFF2025032097000009.tif25170, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain of the anti-CD79b comprises the amino acid sequence of SEQ ID NO: 9, the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, and p is a value of about 2 to about 5 (e.g., about 3.5). In some embodiments, the lyophilized composition is a lyophilized cake. In some embodiments, the lyophilized composition (e.g., a cake) is contained within a vial, e.g., a 20 ml glass vial.

[0049] Further provided is a lyophilized pharmaceutical composition comprising about 30 mg polatuzumab vedotin, about 1.8 mg polysorbate 20, about 1.77 mg succinic acid, about 0.816 mg sodium hydroxide, and about 61.8 mg sucrose. In some embodiments, the lyophilized composition (such as a cake) is contained within a vial, for example, a 20 ml glass vial.

[0050] In some embodiments, the lyophilized pharmaceutical compositions provided herein are lyophilized cakes. In some embodiments, the lyophilized pharmaceutical compositions according to any of the embodiments herein are stable for at least 6, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months upon storage at about 2° C. to about 8° C.

[0051] A liquid pharmaceutical composition is provided comprising a) about 5-60 mg / ml polatuzumab vedotin, b) about 10-200 mM sodium succinate, c) about 100-260 mM sucrose, and d) about 0.06-0.12% by weight / volume polysorbate 20, wherein the liquid composition has a pH of 5-6. In some embodiments, the liquid pharmaceutical composition comprises a) about 10-55 mg / ml polatuzumab vedotin, b) about 10-100 mM sodium succinate, c) about 150-260 mM sucrose, and d) about 0.08-0.12% by weight / volume polysorbate 20, wherein the liquid composition has a pH of 5.1-5.6. In some embodiments, the liquid pharmaceutical composition comprises a) about 15-40 mg / ml polatuzumab vedotin, b) about 10-50 mM sodium succinate, c) about 200-260 mM sucrose, and d) about 0.1-0.12% by weight / volume polysorbate 20, and the pH of the liquid composition is 5.2-5.4. In some embodiments, the liquid composition is obtained by reconstituting a lyophilized composition (such as a cake). In some embodiments, the liquid composition is contained in a vial, for example, a 20 ml glass vial.

[0052] Also provided is a liquid pharmaceutical composition comprising a) 20 mg / ml polatuzumab vedotin, b) 10 mM sodium succinate, c) 120 mM sucrose, d) 0.12% by weight / volume polysorbate 20, wherein the pH of the liquid composition is about 5.3. In some embodiments, the liquid composition is obtained by reconstituting a lyophilized composition (such as a cake). In some embodiments, the liquid composition is contained in a vial, for example, a 20 ml glass vial.

[0053] In some embodiments, the liquid composition according to any of the embodiments herein is stable for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at about 30° C. In some embodiments, the liquid composition according to any of the embodiments herein is stable for at least 24, 48, or 72 hours when stored at about 2° C. to about 8° C. In some embodiments, the liquid composition is obtained by reconstituting a lyophilized composition (such as a cake). In some embodiments, the liquid composition is contained in a vial, for example, a 20 ml glass vial.

[0054] In some embodiments, the reconstituted pharmaceutical compositions provided herein are stable after 72 hours of storage at 2° C.-8° C. In some embodiments, the reconstituted pharmaceutical compositions provided herein are stable after 24 hours of storage at 30° C. with exposure to ambient light. In some embodiments, the reconstituted pharmaceutical composition comprises 20 mg / mL polatuzumab vedotin in 10 mM succinic acid, 120 mM sucrose, and 1.2 mg / mL polysorbate 20, at a pH of 5.3. In some embodiments, the affinity of polatuzumab vedotin to its target (i.e., CD79b) or the biological activity of polatuzumab vedotin.

[0055] In one aspect, the disclosure provides a method of treating a proliferative disorder in a patient in need of such treatment, comprising administering to the patient a pharmaceutical composition or liquid composition as described herein. Also provided is the use of a pharmaceutical composition or liquid composition as described herein for the manufacture of a medicament for treating a proliferative disorder in a patient in need of such treatment. In some embodiments, a pharmaceutical composition or liquid composition as described herein is provided for use in treating a proliferative disorder in a patient in need of such treatment. Also provided is a pharmaceutical composition or liquid composition as described herein for use in a method of treating a proliferative disorder in a patient in need of such treatment.

[0056] In some embodiments, the proliferative disorder is cancer. In an exemplary embodiment, the cancer is a B cell proliferative disorder. In certain embodiments, the B cell proliferative disorder is selected from the group consisting of lymphoma, myeloma, non-Hodgkin's 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 lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0057] In one embodiment of the present disclosure, the B cell proliferative disorder is non-Hodgkin's 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). [Brief description of the drawings]

[0058] [Figure 1] Depict the increase in high molecular weight species (HMWS) after 22 hours of static storage at 30°C at a fixed concentration of PS20 ([PS20]) in three different intravenous (IV) dosing bag sizes. [Diagram 2] Depict the increase in HMWS after 2 hours of agitation stress (100 rpm) at 30°C with a fixed concentration of PS20 ([PS20]) in three different IV dosing bag sizes. [Diagram 3] Depict the effect of temperature on the physical stability of anti-CD79b-vc-MMAE in a saline IV dosing bag at various concentrations of PS20 ([PS20]). [Figure 4]The effect of surfactant and injection fluid type upon static storage at 30° C. for 22 hours is depicted. [Figure 5A] Depict the effect of temperature from 2 to 8 °C on the stability of anti-CD79b-vc-MMAE upon agitation. [Figure 5B] 1 depicts the effect of temperature at 25° C. on the stability of anti-CD79b-vc-MMAE upon agitation. [Figure 5C] 1 depicts the effect of temperature at 30° C. on the stability of anti-CD79b-vc-MMAE upon agitation. [Figure 6] 1 depicts the physical stability of anti-CD79b-vc-MMAE using agitation model 1. [Figure 7] 1 depicts the physical stability of anti-CD79b-vc-MMAE using agitation model 2. [Figure 8] 1 depicts the hydrolysis reaction of thiosuccinimide to stabilize immune complexes and prevent their removal. Maleimide removal can be prevented by hydrolysis of the thiosuccinimide bond present on the immune complex. [Figure 9] 1 depicts the effect of pH on acidic charge variant formation at 30° C. at three different time points, 2 weeks, 4 weeks, and 8 weeks. [Figure 10] 1 depicts the effect of pH on basic charge variant formation at 30° C. at three different time points, 2 weeks, 4 weeks, and 8 weeks. [Figure 11] 1 depicts the effect of pH on HMWS formation at 30° C. at three different time points, 2 weeks, 4 weeks, and 8 weeks. [Figure 12] 1 depicts the effect of pH and buffer type on low molecular weight species (LMWS) stressed at 30° C. for 4 weeks. [Figure 13] Depict the stability of lyophilized formulations at 30° C. by size exclusion chromatography (SEC). [Figure 14] Depict the stability of lyophilized formulations at 30° C. by imaging capillary isoelectric focusing (icIEF). [Figure 15]Figures 15A-C depict the appearance of lyophilized cakes with different protein to sucrose ratios. Figure 15A represents 10 mg / ml anti-CD79b-vc-MMAE and 260 mM sucrose. Figure 15B represents 10 mg / ml anti-CD79b-vc-MMAE and 180 mM sucrose. Figure 15C represents 10 mg / ml anti-CD79b-vc-MMAE and 120 mM sucrose. [Figure 16] Figures 16A-B depict the appearance of lyophilized cakes with different protein to sucrose ratios. Figure 16A represents 10 mg / ml anti-CD79b-vc-MMAE and 260 mM sucrose. Figure 16B represents 20 mg / ml anti-CD79b-vc-MMAE and 120 mM sucrose. [Figure 17] Depict the stability of lyophilized anti-CD79b-vc-MMAE drug product stressed at 2-8 °C, 25 °C, and 40 °C to measure HMWS variants by SEC. [Figure 18] Depict the stability of lyophilized anti-CD79b-vc-MMAE drug product stressed at 2-8 °C, 25 °C, and 40 °C to measure HMWS variants by icIEF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The present disclosure provides stable pharmaceutical compositions comprising an anti-CD79b immunoconjugate and a surfactant. The present disclosure also provides methods for using such compositions for the treatment of cancer.

[0060] I. Definition It is to be understood that the present disclosure is not limited to a particular composition or biological system, which can of course vary. It is also to 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 herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "molecule" includes any combination of two or more such molecules, and so forth.

[0061] As used herein, the term "about" refers to the normal range of error for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0062] It is understood that the aspects and embodiments of the present disclosure described herein include aspects and embodiments "comprising," "consisting," and "consisting essentially of."

[0063] The term "anti-CD79b immunoconjugate" refers to an anti-CD79b antibody-drug conjugate (ADC). As used herein, an anti-CD79b immunoconjugate contains 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-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB).

[0064] The term "antibody" is used in the broadest sense and specifically encompasses, for example, single anti-CD79b monoclonal antibodies (including, but not limited to, agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), anti-CD79b antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain anti-CD79b antibodies, as well as fragments (see below) of anti-CD79b antibodies (including Fab, Fab', F(ab')2, and Fv fragments), diabodies, single domain antibodies (sdAbs), so long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody. Antibodies may be chimeric, human, humanized, and / or affinity matured.

[0065] The term "anti-CD79b antibody" or "antibody that binds to CD79b" refers to an antibody that can bind to CD79b with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD79b. Preferably, the extent of binding of an anti-CD79b antibody to unrelated, non-CD79b proteins is less than about 10% of the binding of the antibody to CD79b, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD79b has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certain embodiments, an anti-CD79b antibody binds to an epitope of CD79b that is conserved among CD79bs from different species.

[0066] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies contain 10 antigen-binding sites because they consist of five basic heterotetrameric units together with an additional polypeptide called the J chain, while secretory IgA antibodies can polymerize to form multivalent assemblies containing 2-5 basic four-chain units together with the J chain). For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its opposite end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that certain amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of the VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, page 71 and chapter 6 of Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994.

[0067] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0068] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy chain variable domain may be referred to as "VH". The light chain variable domain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding sites.

[0069] The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions", each 9-12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs that adopt a predominantly β-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as involvement of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0070] An "intact" antibody is one that contains not only an antigen-binding site, but also 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.

[0071] A "naked antibody" for purposes herein is an antibody that is not conjugated to a drug moiety or radiolabel.

[0072] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises the antigen-binding site of an intact antibody and thus retains the ability to bind to an antigen.

[0073] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments and a residual "Fc" fragment (a designation reflecting the ability to crystallize readily). The Fab fragment consists of the entire L chain together with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies results in a single large F(ab')2 fragment, which generally corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and are still capable of cross-linking to antigen. Fab' fragments differ from Fab fragments by having additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0074] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences within the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain cell types.

[0075] An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight non-covalent association. In single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in two-chain Fv species. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0076] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For general reviews on sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995 (infra).

[0077] The term "diabody" refers to an antibody fragment with two antigen-binding sites, these fragments comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Small antibody fragments are prepared by constructing sFv fragments (see preceding paragraphs) with a short linker (about 5-10 residues) between the VH and VL domains, such that interchain, rather than intrachain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Diabodies can be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in detail, for example, in EP 404,097, WO 93 / 11161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0078] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methodologies in bacterial, eukaryotic, or plant cells (see U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

[0079] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0080] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, hypervariable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being FRs of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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: 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) and references cited therein.

[0081] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. The methods described in Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) are also available for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic mice, that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies generated via human B cell hybridoma technology.

[0082] As used herein, the term "hypervariable region", "HVR", or "HV" refers to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several hypervariable region descriptive methods are in use and are encompassed herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions are a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. Residues from each of these hypervariable regions are described below. TIFF2025032097000010.tif74170

[0083] The hypervariable regions may comprise 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. The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions.

[0084] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0085] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), as well as inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the homology regions of the antibody's sequence with the "standard" Kabat numbering sequence.

[0086] The Kabat numbering system is generally used when referring to residues in the variable domain (residues about 1-107 of the light chain and residues 1-113 of the heavy chain) (see, e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in the variable domain of an antibody refers to the residue numbering according to the Kabat numbering system. Unless otherwise stated herein, reference to a residue number in the constant domain of an antibody refers to the residue numbering according to the EU numbering system (see, e.g., U.S. Provisional Patent Application No. 60 / 640,323, figures for EU numbering).

[0087] An "affinity matured" antibody is an antibody that has one or more modifications in one or more of its HVRs, which modifications result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not have these modification(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by techniques known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues has been described by Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994), Schier et al. Gene 169:147-155 (1995), Yelton et al. J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0088] "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 indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Specific exemplary embodiments are described below.

[0089] As used herein to refer to binding affinity, "or better" refers to stronger binding between a molecule and its binding partner. As used herein, "or better" refers to stronger binding represented by a smaller Kd number. For example, in an antibody having an affinity for an antigen of ".6 nM or better," the affinity of the antibody for the antigen is less than .6 nM, i.e., .59 nM, .58 nM, .57 nM, etc., or any value less than .6 nM.

[0090] In one embodiment, "Kd" or "Kd value" according to the present disclosure is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen, described by the following assay, which measures the solution binding affinity of the Fab to the antigen by equilibrating the Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen on an anti-Fab antibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). To establish conditions for this assay, microtiter plates (Dynex) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% bovine serum albumin (w / v) in PBS for 2-5 hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125I] antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is then incubated overnight, although incubation may be continued for a longer period (e.g., 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% Tween-20 in PBS. Once the plate has dried, 150 μl / well of scintillant (MicroScint-20, Packard) is added and the plate is counted in a TOPCOUNT™ gamma counter (Packard) for 10 minutes. Concentrations of each Fab that yielded less than or equal to 20% of maximal binding are selected for use in competitive binding assays.

[0091] According to another embodiment, "Kd" or "Kd value" is measured using a surface plasmon resonance assay using a BIAcore™ 2000 or BIAcore™ 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5ug / ml (approximately 0.2uM) with 10mM sodium acetate (pH 4.8) and then injected at a flow rate of 5ul / min to achieve approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at a flow rate of approximately 25 ul / min at 25° C. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the binding rate by the surface plasmon resonance assay described above is 10 6 M -1 S -1Above 100 nm, the binding rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations as measured with a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) equipped with a stirred red cuvette.

[0092] "Binding rate" or "rate of association" or "association rate" or "k on " can also be determined using the same surface plasmon resonance techniques described above, using the BIAcore™ 2000 or BIAcore™ 3000 (BIAcore, Inc., Piscataway, NJ).

[0093] As used herein, the phrases "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two numerical values ​​(typically one associated with an antibody of the present disclosure and the other associated with a reference / comparator antibody) such that one of skill in the art would regard the difference between the two values ​​as having little or no biological and / or statistical significance in the context of the biological characteristic measured by the values ​​(e.g., Kd values). 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%, preferably less than about 10% as a function of the value for the reference / comparator antibody.

[0094] As used herein, the phrases "substantially reduced" or "substantially different" refer to a sufficiently high degree of difference between two numerical values ​​(typically one associated with an antibody of the present disclosure and the other associated with a reference / comparator 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). The difference between the two values ​​is preferably more than about 10%, preferably more than about 20%, preferably more than about 30%, preferably more than about 40%, preferably more than about 50% as a function of the value for the reference / comparator antibody.

[0095] An "acceptor human framework" for purposes herein is a framework that comprises the amino acid sequence of a VL framework or a VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence as that of the human immunoglobulin framework or may contain pre-existing amino acid sequence changes. If pre-existing amino acid changes are present, preferably there are no more than 5, and preferably no more than 4 or no more than 3 pre-existing amino acid changes. If pre-existing amino acid changes are present in the VH, preferably those changes are at only 3, 2, or 1 of positions 71H, 73H, and 78H, e.g., the amino acid residues at those positions may be 71A, 73T, and / or 78A. In one embodiment, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0096] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al.

[0097] A "VH subgroup III consensus framework" comprises a consensus sequence obtained from amino acid sequences within variable heavy subgroup III of Kabat et al. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each 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).

[0098] A "VL subgroup I consensus framework" comprises a consensus sequence obtained from amino acid sequences within the variable light kappa subgroup I of Kabat et al. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least a portion or all of each 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).

[0099] An "unmodified human framework" is a human framework that has the same amino acid sequence as an acceptor human framework, e.g., lacks the human to non-human amino acid substitution(s) in the acceptor human framework.

[0100] An anti-CD79b immune complex or an antibody that "binds" to CD79b is an antibody that binds to the CD79b antigen with sufficient affinity so that the antibody is useful as a therapeutic in targeting cells or tissues expressing the antigen and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein is less than about 10% of the binding of the antibody to its specific target protein, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With respect to the binding of an antibody to a target molecule, the term "specific binding" to or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule in comparison to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, for example, an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the term "specific binding" to or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target refers to, for example, a binding that is at least about 10 times more specific to 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 -11 M, or at least about 10 -12M, or greater. In one embodiment, the term "specific binding" refers to binding by an antibody to an epitope on a CD79b polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0101] An "effector function" of an antibody refers to a biological activity attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0102] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population with all K447 residues removed, an antibody population with no K447 residue removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0103] A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require the combination of an Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays disclosed in the definitions herein.

[0104] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region (non-A and A allotypes), native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0105] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide, compared to the native sequence Fc region or the Fc region of the parent polypeptide. A variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0106] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "equip" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells for mediating ADCC, 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, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).

[0107] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds an IgG antibody (gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See review in M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, 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). Other FcRs, including those yet to be identified, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0108] The in vivo binding to human FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with variant Fc regions are administered. WO2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al. J.Biol.Chem.9(2):6591-6604 (2001).

[0109] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from a natural source, e.g., blood.

[0110] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind their cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity (polypeptides with variant Fc regions) are described, for example, in U.S. Pat. No. 6,194,551 B1 and WO 1999 / 51642. See, for example, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0111] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinantly engineering the nucleic acid encoding the antibody. Thus, a composition comprising an antibody with an Fc region according to the present disclosure may include an antibody with K447, an antibody with all K447 removed, or a mixture of antibodies with and without the K447 residue.

[0112] A "B cell surface marker" or "B cell surface antigen" herein is an antigen expressed on the surface of a B cell that can be targeted with an antagonist that binds to the B cell, including, but not limited to, an antibody to a B cell surface antigen or a soluble form of a B cell surface antigen that can attenuate the binding of a ligand to the naturally occurring B cell antigen. 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 leukocyte surface markers (for a description see The Leukocyte Antigen Facts Book, 2nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & 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. B cell surface markers of particular interest are preferentially expressed on B cells compared to other non-B cell tissues in mammals, and may be expressed on both precursor and mature B cells.

[0113] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, hematopoietic or blood-related cancers, such as lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as splenic and lymph node cancers, as well as carcinomas, blastomas, and sarcomas. More specific examples of cancers include, for example, high-, intermediate-, and low-grade lymphomas (including B-cell lymphomas such as mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin's lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and Hodgkin's lymphoma, as well as T-cell lymphomas), and B-cell-related cancers including leukemias (including secondary leukemias, chronic lymphocytic leukemia (CLL) (such as B-cell leukemia (CD5+ B lymphocytes)), myeloid leukemias (such as acute myeloid leukemia, chronic myeloid leukemia), lymphocytic leukemias (such as acute lymphoblastic leukemia (ALL)), and myelodysplasia), as well as other hematological and / or B- or T-cell-related cancers. Also included are cancers of additional hematopoietic cells, including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils, and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells. Also included are cancerous B-cell proliferative disorders selected from the following: lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), DLBCL, relapsed / refractory DLBCL, FL, and mantle cell lymphoma. The origins of B cell cancers include: marginal zone B cell lymphoma arises within memory B cells in the marginal zone, follicular lymphoma and diffuse large B cell lymphoma arise within the central cells of the luminal zone of the germinal center, chronic lymphocytic leukemia and small lymphocytic leukemia arise within B1 cells (CD5+), mantle cell lymphoma arises within naive B cells in the mantle zone, and Burkitt's lymphoma arises within the centroblasts of the luminal zone of the germinal center.Tissues containing hematopoietic cells, referred to herein as "hematopoietic cell tissues", include the thymus and bone marrow, as well as peripheral lymphoid tissues, such as the spleen, lymph nodes, lymphoid tissues associated with mucosa, such as gut-associated lymphoid tissue, tonsils, Peyer's patches, and appendix, as well as lymphoid tissues associated with other mucosa, such as the bronchial lining. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, carcinoma of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, small intestine cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0114] As used herein, "B cell malignancies" or "B cell proliferative disorders" include low-grade / follicular NHL, small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, non-Hodgkin's lymphoma (NHL), DLBCL, relapsed / refractory NHL, and other types of B-cell malignancies. Non-Hodgkin's lymphoma (NHL), including recurrent DLBCL, FL, lymphocyte-predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), and indolent NHL (including relapsed indolent NHL and rituximab-refractory indolent NHL); leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia; mantle cell lymphoma; and other hematological malignancies. Such malignancies can be treated with antibodies directed against B-cell surface markers, such as CD79b. Such diseases are contemplated herein to be treated by administration of antibodies directed against B-cell surface markers, such as CD79b, including administration of unconjugated ("naked") antibodies or antibodies conjugated to the cytotoxic agents disclosed herein. It is also contemplated herein that such diseases are treated by combination therapy, including an anti-CD79b antibody or anti-CD79b antibody-drug conjugate of the present disclosure in combination with another antibody or antibody-drug conjugate, another cytotoxic agent, radiation or other treatment, administered simultaneously or sequentially. In an exemplary treatment method of the present disclosure, an anti-CD79b antibody of the present disclosure is administered in combination with an anti-CD20 antibody, immunoglobulin, or CD20-binding fragment thereof, either together or sequentially. The anti-CD20 antibody can be a naked antibody or an antibody-drug conjugate. In one embodiment of the combination therapy, the anti-CD79b antibody is an antibody of the present disclosure and the anti-CD20 antibody is Rituxan® (rituximab).

[0115] As used herein, the term "non-Hodgkin's lymphoma" or "NHL" refers to a cancer of the lymphatic system other than Hodgkin's lymphoma. Hodgkin's lymphomas can generally be distinguished from non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and the absence of such cells in non-Hodgkin's lymphomas. As used herein, examples of non-Hodgkin's lymphomas encompassed by this term include all those identified as such by a skilled artisan (e.g., an oncologist or pathologist) according to classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme set forth in Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). See in particular the listings in Figures 11.57, 11.58, and 11.59.More specific examples include relapsed or refractory NHL, frontline low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia 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, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular lymphoma) These include, but are not limited to, aggressive diffuse NHL, diffuse large B-cell lymphoma, relapsed / refractory diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), NHL relapsed after or refractory to autologous stem cell transplant, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, skin (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0116] A "disorder" is any condition that may benefit from treatment with the substance / molecule or method of the present disclosure. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders that may be treated herein include cancerous conditions, such as malignant and benign tumors; non-leukemia and lymphoid tumors; neuronal disorders, glial disorders, astrocytic disorders, hypothalamic and other glandular disorders, macrophage disorders, epithelial disorders, stromal disorders, blastocoelic disorders; and inflammatory disorders, immune disorders, and other angiogenesis-related disorders. Disorders further include cancerous conditions such as B cell proliferative disorders and / or B cell tumors, e.g., lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0117] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer. In some embodiments, the cancer is a B-cell proliferative disorder.

[0118] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0119] The term "treat" or "treatment" or "palliative" refers to both therapeutic and prophylactic or preventative treatments, the purpose of which is to prevent or slow down (alleviate) the targeted pathological condition or disorder. Those in need of treatment include those who already have the disorder, as well as those who are prone to develop the disorder or who need to prevent the disorder. A subject or mammal is successfully "treated" of a CD79b polypeptide-expressing cancer if, after receiving a therapeutic amount of an anti-CD79b antibody according to the methods of the present disclosure, the patient shows 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; an inhibition (i.e., slowed to some extent and preferably stopped) of cancer cell invasion into peripheral organs, including the spread of cancer to soft tissues and bones; an inhibition (i.e., slowed to some extent and preferably stopped) of tumor metastasis; an inhibition (i.e., slowed to some extent and preferably stopped) of tumor growth; and / or a reduction to some extent of one or more of the symptoms associated with the particular cancer; a reduction in morbidity and mortality, and an improvement in quality of life issues. To the extent the anti-CD79b antibody may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic, and the reduction of these signs or symptoms may also be felt by the patient.

[0120] The above parameters for evaluating the success of treatment and improvement of disease can be easily measured by routine procedures known to physicians. In cancer therapy, efficacy can be measured, for example, by evaluating the time to disease progression (TTP) and / or determining the response rate (RR). Metastasis can be determined by staging tests, by bone scans and by testing calcium levels and other enzymes to determine spread to bone. CT scans may be performed to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastases to the lungs and liver, respectively. Other routine methods for monitoring disease include transrectal ultrasound (TRUS) and transrectal needle biopsy (TRNB).

[0121] In the case of bladder cancer, a more localized cancer, methods for determining disease progression include urinary cytological evaluation by cystoscopy, monitoring for the presence of blood in the urine, visualization of the urothelial ducts by ultrasound or intravenous pyelography, computed tomography (CT), and magnetic resonance imaging (MRI). The presence of distant metastases can be assessed by abdominal CT, chest x-ray, or skeletal radionuclide imaging.

[0122] "Chronic" administration refers to administration of an agent(s) in a continuous manner such that the initial therapeutic effect (activity) is maintained over an extended period of time, as opposed to an acute manner. "Intermittent" administration is treatment that is not continuous without interruption, but rather cyclic in nature.

[0123] An "individual" or "subject" or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal is a human.

[0124] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0125] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed at the dosages and concentrations used. In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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 non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0126] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation of an anti-CD79b immunoconjugate in a form such that the biological activity of the active ingredient is effective and does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective dosage of the active ingredient employed.

[0127] A "sterile" preparation is aseptic or free or essentially free of all viable microorganisms and their spores.

[0128] An "effective amount" of an anti-CD79b immunoconjugate disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner for the stated purpose.

[0129] The term "therapeutically effective amount" refers to an amount of an anti-CD79b immunoconjugate effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of the immunoconjugate can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with cancer. See the definition of "treat" herein. To the extent the immunoconjugate can prevent the growth of and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. A "prophylactically effective amount" refers to an amount effective, at the dosages and duration required, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount will be less than a therapeutically effective amount, since a prophylactic dose is used in a subject prior to or at an earlier stage of the disease.

[0130] A "growth inhibitory amount" of an anti-CD79b immunoconjugate refers to an amount capable of inhibiting the growth of a cell, particularly a tumor (e.g., a cancer cell), either in vitro or in vivo. A "growth inhibitory amount" of an anti-CD79b immunoconjugate for purposes of inhibiting neoplastic cell growth can be determined empirically and in a routine manner.

[0131] A "cytotoxic amount" of an anti-CD79b immunoconjugate refers to an amount capable of causing destruction of a cell, particularly a tumor (e.g., cancer cell), either in vitro or in vivo. A "cytotoxic amount" of an anti-CD79b antibody for purposes of inhibiting neoplastic cell growth can be determined empirically and in a routine manner.

[0132] A "CD79b-expressing cell" is a cell that expresses endogenous or transfected CD79b polypeptide, either on the cell surface or in a secreted form. A "CD79b-expressing cancer" is a cancer that includes cells in which CD79b polypeptide is present on the cell surface or that produce or secrete CD79b polypeptide. A "CD79b-expressing cancer" is optionally one that produces sufficient levels of CD79b polypeptide on its cell surface such that an anti-CD79b antibody can bind to it and have a therapeutic effect on the cancer. In another embodiment, a "CD79b-expressing cancer" is optionally one that produces and secretes sufficient levels of CD79b polypeptide such that an anti-CD79b antibody antagonist can bind to it and have a therapeutic effect on the cancer. With regard to the latter, the antagonist can be an antisense oligonucleotide that can reduce, inhibit, or prevent the production and secretion of secreted CD79b polypeptide by tumor cells. A cancer that "overexpresses" a CD79b polypeptide is one that has higher levels of CD79b polypeptide on its cell surface or produces and secretes it compared to non-cancerous cells of the same tissue type. Such overexpression can be caused by gene amplification or by increased transcription or translation. Overexpression of a CD79b polypeptide can be determined in a detection or prognostic assay by assessing increased levels of CD79b protein present on the cell surface or secreted by the cell (e.g., via immunohistochemistry assays, FACS analysis, etc., using an anti-CD79b antibody prepared against an isolated CD79b polypeptide, which can be prepared from an isolated nucleic acid encoding the CD79b polypeptide using recombinant DNA technology).Alternatively, or in addition, levels of nucleic acid or mRNA encoding a CD79b polypeptide in cells may be measured, for example, via fluorescent in situ hybridization (FISH, see WO 98 / 45479 published October 1998), Southern blot, Northern blot, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR) using nucleic acid-based probes corresponding to a nucleic acid encoding CD79b or its complement. For example, CD79b polypeptide overexpression can also be studied by measuring shed antigens in biological fluids such as serum using antibody-based assays (see, e.g., U.S. Pat. No. 4,933,294, issued June 12, 1990; WO 91 / 05264, issued April 18, 1991; U.S. Pat. No. 5,401,638, issued March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). Apart from the above assays, various in vivo assays are available to the skilled practitioner. For example, cells in the patient's body can be exposed to an antibody, optionally labeled with a detectable label (e.g., a radioisotope), and binding of the antibody to cells in the patient can be assessed, for example, by external scanning for radioactivity or by analysis of a biopsy taken from a patient previously exposed to the antibody.

[0133] As used herein, the term "cytotoxic agent" refers to a drug molecule that inhibits or prevents cell function and / or causes destruction of cells. The term is intended to include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof that may have deleterious effects on cell growth or proliferation. In one embodiment, the cytotoxic agent is mono-methyl auristatin E (MMAE).

[0134] As used herein, a "lyophilized" composition refers to a liquid composition that has been subjected to freeze-drying via the process of lyophilization, resulting in a "cake". In certain embodiments, the freeze-dried cake is stable under storage conditions (5°C ± 3°C and protected from light) without significant changes in cake structure, color, appearance, or moisture content. In a particular embodiment, the freeze-dried cake is smooth without creases. In certain embodiments of the present disclosure, the freeze-dried composition is stable for about 60 months. In certain embodiments of the present disclosure, the freeze-dried composition is stable for about 48 months.

[0135] A "stable" formulation is one in which the immunoconjugate therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be assessed qualitatively and / or quantitatively in a variety of different ways, for example, by assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection), by assessing charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary zone electrophoresis, by amino- or carboxy-terminal sequence analysis, by mass spectrometry, by SDS-PAGE analysis to compare reduced and intact antibodies, by peptide mapping (e.g., trypsin or LYS-C) analysis, by assessing the biological activity or antigen binding function of the antibody, etc. Instability can involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0136] An immunoconjugate "retains its physical stability" in a pharmaceutical formulation if it shows no or little signs of aggregation, precipitation, and / or denaturation upon visual inspection for color and / or clarity, or as measured by UV light scattering or size exclusion chromatography.

[0137] An immunoconjugate "retains its chemical stability" in a pharmaceutical formulation if its chemical stability at a given time is such that the immunoconjugate is considered to still retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying the protein moiety of the chemically modified form of the immunoconjugate (e.g., an antibody). Chemical modification can involve size modification (e.g., clipping), which can be assessed, for example, using size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical modification include protein charge modification (e.g., resulting from deamidation), which can be assessed, for example, by ion exchange chromatography or icIEF. Additionally or alternatively, the chemical stability of the immunoconjugate can be assessed by detecting and quantifying the drug moiety of the chemically modified form of the immunoconjugate. Additionally or alternatively, the chemical stability of the immunoconjugate can be assessed, for example, by measuring the drug:antibody ratio (DAR) via hydrophobic interaction chromatography (HIC) to determine the DAR distribution in a composition comprising the immunoconjugate.

[0138] An antibody of an immunoconjugate "retains its biological activity" in a pharmaceutical formulation if the biological activity of the protein portion of the immunoconjugate (e.g., an antibody such as an anti-CD79b antibody) at a given time, as determined in an assay (e.g., an antigen binding assay), is at least about 60% (within the error of the assay) of the biological activity exhibited at the time the pharmaceutical formulation containing the immunoconjugate was prepared.

[0139] A "deamidated" monoclonal antibody herein is one in which one or more asparagine residues have been derivatized, eg, to aspartic acid or isoaspartic acid.

[0140] An "oxidized" monoclonal antibody herein is an antibody in which one or more tryptophan residues and one or more methionines thereof have been oxidized.

[0141] A "glycosylated" monoclonal antibody herein is an antibody in which one or more lysine residues are glycosylated.

[0142] An antibody that is "susceptible to deamidation" is one that contains one or more residues that are found to be susceptible to deamidation.

[0143] An antibody that is "susceptible to oxidation" is one that contains one or more residues that are found to be susceptible to oxidation.

[0144] An antibody that is "prone to aggregation" is one that is found to aggregate with other antibody molecule(s), particularly upon freezing and / or agitation.

[0145] An antibody that is "susceptible to fragmentation" is one that is found to be cleaved into two or more fragments, for example, at its hinge region.

[0146] "Reducing deamidation, oxidation, aggregation, or fragmentation" is intended to prevent or reduce the amount of deamidation, oxidation, aggregation, or fragmentation compared to a monoclonal antibody formulated in a different formulation.

[0147] The antibodies that are formulated are preferably essentially pure and desirably essentially homogeneous (e.g., free from contaminating proteins, etc.). An "essentially pure" antibody refers to a composition that comprises at least about 90% by weight, preferably at least about 95% by weight, of antibody, based on the total weight of protein in the composition. An "essentially homogeneous" antibody refers to a composition that comprises at least about 99% by weight, of antibody, based on the total weight of protein in the composition.

[0148] "Isotonic" means that the formulation has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsm. Isotonicity can be measured, for example, using a vapor pressure osmometer or a freeze osmometer.

[0149] As used herein, a "buffer" refers to a buffer solution that resists changes in pH due to the action of its acid-base complex components. Non-limiting examples of buffers herein include histidine, sodium phosphate, and sodium succinate. The buffers of the present 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 an exemplary embodiment, the buffer has a pH of 5.3. For example, sodium succinate is an example of a buffer that controls the pH within this range.

[0150] As used herein, "surfactant" refers to a surface active agent, preferably a non-ionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl beta amine, or cetyl betaine; lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol, and ethylene glycol-propylene glycol copolymer (e.g., Pluronics, PF68, etc.). In one embodiment, the surfactant herein is polysorbate 20. In another embodiment, the surfactant herein is polysorbate 80.

[0151] As used herein, "sugar" refers to a soluble carbohydrate. Non-limiting examples of sugars include glucose, fructose, sucrose, trehalose, arginine, glycerin, proline, dextran, and sugar alcohols (such as glycerol, mannitol, and sorbitol).

[0152] II. Pharmaceutical Compositions In one aspect, the disclosure provides a pharmaceutical composition comprising an anti-CD79b immunoconjugate and a surfactant, wherein the surfactant is at a concentration of at least 0.06% weight / volume (i.e., 0.6 mg / ml), and the anti-CD79b immunoconjugate has the formula: TIFF2025032097000011.tif25170, wherein The pharmaceutical composition is provided wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p is a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5).

[0153] In some embodiments, the composition disclosed herein is lyophilized.In some embodiments, the composition disclosed herein is a reconstituted composition, i.e., a composition reconstituted from a lyophilized cake.In some embodiments, a 20ml glass vial (such as a sealed vial) is provided that contains the pharmaceutical composition described herein (e.g., a lyophilized composition or a reconstituted composition).

[0154] A. Immune complex The present disclosure relates to immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising an antibody conjugated to a small molecule toxin, such as monomethylauristatin (MMAE), a synthetic analog of dolastatin. The present disclosure provides an anti-CD79b immunoconjugate of the formula Ab-(LD)p, where Ab is an anti-CD79b antibody, the anti-CD79b antibody comprising a heavy chain and a light chain, the light chain comprising (a) the HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO:1), (b) the HVR-L2 sequence of AASNLES (SEQ ID NO:2), and (c) the HVR-L3 sequence of QQSNEDPLT (SEQ ID NO:3), and the heavy chain comprising (a) the HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO:4), (b) the HVR-L2 sequence of AASNLES (SEQ ID NO:5), and (c) the HVR-L3 sequence of QQSNEDPLT (SEQ ID NO:6). and (c) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (sequence number 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (sequence number 6), wherein L is a linker comprising 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB), D is MMAE, and p is a value of about 1 to about 8 (about 2 to about 5, for example, about 3.5), and the anti-CD79b immune complex has the following structure: TIFF2025032097000012.tif24170Anti-CD79b immunoconjugates of the present disclosure may be referred to as anti-CD79b-MC-val-cit-MMAE, anti-CD79b-MC-vc-MMAE, or anti-CD79b-vc-MMAE. Polatuzumab vedotin is an example of an anti-CD79b immunoconjugate of the present disclosure. Polatuzumab vedotin has CAS Registry Number 1313206-42-6, IUPHAR / BPS Number 8404, and KEGG Number D10761. "Polatuzumab vedotin", "DCDS4501A", and "RG7596" encompass all corresponding immunoconjugates that meet the requirements necessary to obtain approval for promotion as an identical or biosimilar product in a country or region selected from the group of countries consisting of the United States, Europe, and Japan.

[0155] In some embodiments, the anti-CD79b immune complex is at a concentration in the pharmaceutical composition of 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 a specific embodiment, the anti-CD79b immunoconjugate is at a concentration of 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 in the pharmaceutical composition.

[0156] In some embodiments of any of the embodiments provided herein, a "about" value or parameter encompasses an error range of any one of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value or parameter, including any ranges therebetween.

[0157] In some embodiments, the anti-CD79b immune complex is at a concentration of about 10 mg / ml to about 20 mg / ml in the pharmaceutical composition. In an exemplary embodiment, the anti-CD79b immune complex is at a concentration of about 10 mg / ml in the pharmaceutical composition. In another exemplary embodiment, the anti-CD79b immune complex is at a concentration of about 20 mg / ml in the pharmaceutical composition.

[0158] In some embodiments, the anti-CD79b immune complex is at a concentration of 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 the pharmaceutical composition. In a specific embodiment, the anti-CD79b immune complex is at a concentration of 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 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 in the pharmaceutical composition.

[0159] In some embodiments, the anti-CD79b immune complex is at a concentration of 10 mg / ml to 20 mg / ml in the pharmaceutical composition. In an exemplary embodiment, the anti-CD79b immune complex is at a concentration of 10 mg / ml in the pharmaceutical composition. In another exemplary embodiment, the anti-CD79b immune complex is at a concentration of 20 mg / ml in the pharmaceutical composition.

[0160] As contemplated herein, an anti-CD79b immunoconjugate is of formula I, as shown below, in which an anti-CD79b antibody or fragment thereof (Ab) is conjugated (i.e., covalently attached) to an MMAE drug moiety (D) through a linker (L). Ab-(LD) p I

[0161] In formula I, p is the average number of drug moieties per antibody, which can range, for example, from about 1 to about 20 drug moieties per antibody, and in certain embodiments, from 1 to about 8 drug moieties per antibody. The disclosure includes compositions comprising antibody-drug compound mixtures of formula I, wherein the average drug loading per antibody is about 2 to about 5 or about 3 to about 4, e.g., 3.5.

[0162] 1. Anti-CD79b antibody The present disclosure provides immunoconjugates comprising an anti-CD79b antibody, or a functional fragment thereof.

[0163] In one aspect, the disclosure provides an anti-CD79b antibody that 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 diabody, 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 respective antigen epitope. The antibodies of the disclosure are optionally produced in CHO cells or bacterial cells, and are preferably capable of inducing the death of cells to which they bind.

[0164] The present disclosure provides an anti-CD79b immunoconjugate comprising a humanized anti-CD79b antibody, wherein the monovalent affinity of the antibody to CD79b (e.g., the affinity of the antibody as a Fab fragment to CD79b) is substantially the same as the monovalent affinity of a murine antibody (e.g., the affinity of the murine antibody as a Fab fragment to CD79b) or a chimeric antibody (e.g., the affinity of the chimeric antibody as a Fab fragment to CD79b).

[0165] In one embodiment, the disclosure provides an anti-CD79b immune complex comprising a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form to CD79b (e.g., the affinity of the antibody to CD79b as an IgG) is 0.3 nM or better. In another embodiment, the disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form to CD79b (e.g., the affinity of the antibody to CD79b as an IgG) is 0.5 nM. In a further embodiment, the disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form to CD79b (e.g., the affinity of the antibody to CD79b as an IgG) is 0.5 nM+ / -0.1. In another embodiment, the disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form to CD79b (e.g., the affinity of the antibody to CD79b as an IgG) is 0.3 nM-0.7 nM. In another embodiment, the disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form to CD79b (e.g., the affinity of the antibody to CD79b as an IgG) is between 0.4 nM and 0.6 nM. In another embodiment, the disclosure provides a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form to CD79b (e.g., the affinity of the antibody to CD79b as an IgG) is between 0.5 nM and 0.55 nM.

[0166] As is well established 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 terms of a variety of quantitative values. Thus, in one embodiment, the binding affinity is expressed as a Kd value, which reflects the intrinsic binding affinity (e.g., with minimized avidity effects). Typically and preferably, the binding affinity is measured in vitro, either in a cell-free or cell-associated setting. As described in more detail herein, the fold difference in binding affinity can be quantified in terms of the ratio of the monovalent binding affinity value of a humanized antibody (e.g., in Fab form) to the monovalent binding affinity value of a reference / comparator antibody (e.g., a murine antibody with donor hypervariable region sequences) (e.g., in Fab form), where these binding affinity values ​​are determined under similar assay conditions. Thus, in one embodiment, the fold difference in binding affinity is determined as the ratio of the Kd value of a humanized antibody in Fab form to the Kd value of the reference / comparator Fab antibody. For example, in one embodiment, if an antibody (A) of the disclosure has an affinity that is "3-fold lower" than that of a reference antibody (M), then the Kd value of A is 3-fold, the Kd value of M is 1-fold, and the ratio of Kd of A to Kd of M is 3:1. Conversely, in one embodiment, if an antibody (C) of the disclosure has an affinity that is "3-fold greater" than that of a reference antibody (R), then the Kd value of C is 1-fold, the Kd value of R is 3-fold, and the ratio of Kd of C to Kd of R is 1:3. Binding affinity measurements can be obtained using any of a number of assays known in the art, including those described herein, including, for example, Biacore, radioimmunoassay (RIA), and ELISA.

[0167] The anti-CD79b antibodies provided comprise a heavy chain and a light chain, wherein the light chain comprises (a) the HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) the HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) the HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), and the heavy chain comprises (a) the HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) the HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) the HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6).

[0168] In some embodiments of the disclosure, the anti-CD79b antibody is EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSS (SEQ ID NO: 7) A heavy chain variable domain (VH) comprising the amino acid sequence of The light chain variable domain (VL) comprises the amino acid sequence of DIQLTQSPSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIKR (SEQ ID NO: 8).

[0169] In some embodiments, the heavy chain comprises: EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9) and the light chain comprises the amino acid sequence It comprises the amino acid sequence of DIQLTQSPSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0170] 2. Linker The present disclosure provides linkers that include several moieties, including 6-maleimidocaproyl ("MC"), valine-citrulline ("val-cit" or "vc"), and p-aminobenzyloxycarbonyl ("PAB"). Such linker moieties are known in the art and are briefly described below.

[0171] As used herein, a linker has the following formula II: TIFF2025032097000013.tif12170

[0172] where A is a Stretcher unit, a is an integer between 0 and 1, W is an amino acid unit, w is an integer between 0 and 12, Y is a Spacer unit, and y is 0, 1, or 2. A "Stretcher unit" links an antibody to another linker component. Stretcher units MC provided herein are shown below (where the wavy line indicates the site of covalent attachment to the antibody): TIFF2025032097000014.tif29170

[0173] The amino acid unit allows for cleavage of the linker by proteases, thereby facilitating the release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21:778-784. The amino acid unit may include naturally occurring amino acid residues, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsin B, C, and D, or plasmin proteases. The amino acid unit provided herein is a valine-citrulline (vc or val-cit) dipeptide.

[0174] As used herein, a "spacer" unit is self-immolative and links the antibody to the drug moiety via the stretcher unit and the amino acid unit. A "self-immolative" spacer unit allows the release of the drug moiety without a separate hydrolysis step as a result of an amide bond, where a carbamate, methylcarbamate, or carbonate is made between benzyl alcohol and a cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103. A spacer unit provided herein is p-aminobenzyloxycarbonyl (PAB).

[0175] Linker components, including Stretcher units, Spacer units, and Amino Acid units, can be synthesized by methods known in the art, such as those described in US 2005 / 0238649(A1).

[0176] The linker of the present disclosure is MC-val-cit-PAB, shown below. TIFF2025032097000015.tif42170

[0177] 3. Monomethylauristatin (MMAE) The immunoconjugates of the present disclosure include an anti-CD79b antibody or fragment thereof conjugated to an auristatin, a synthetic analog of dolastatin (US Pat. Nos. 5,635,483 and 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer activity (US Pat. No. 5,663,149) and antibacterial activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The auristatin drug moiety can be attached to the antibody through the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172). The auristatins of the disclosure are synthetic, uncharged, N-terminally linked monomethylauristatin (MMAE) pentapeptides, shown below. TIFF2025032097000016.tif26170

[0178] Anti-CD79b immunoconjugates of formula I comprising an anti-CD79b antibody or fragment thereof, a linker with various linker components, and MMAE are shown below with the following abbreviations (where "Ab" is an anti-CD79b antibody or fragment thereof, p is 1 to about 8 (e.g., about 2 to about 5, such as about 3.5), "Val-Cit" or "vc" is a valine-citrulline dipeptide, and "S" is a sulfur atom): TIFF2025032097000017.tif30170

[0179] It should be noted that in certain structural descriptions of sulfur-linked immunoconjugates herein, the antibody is represented as "Ab-S" not to indicate that a particular sulfur atom carries multiple linker-drug moieties, but simply to indicate the nature of the sulfur linkage. The left bracket in the following structure may also be placed to the left of the sulfur atom between Ab and S, which is an equivalent description of the immunoconjugates of the present disclosure described herein.

[0180] 4. Drug Loading Drug loading is represented by p, the average number of drug moieties per antibody in a molecule of Formula I. Drug loading can range from 1 to 20 drug moieties (D) per antibody. ADCs of Formula I include a collection of antibodies conjugated to drug moieties ranging from 1 to 20. The average number of drug moieties per antibody when preparing an ADC from a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of ADCs with respect to p can also be determined. In some cases, separation, purification, and characterization of homogenous ADCs with a particular value of p from ADCs with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis. Thus, pharmaceutical formulations of antibody-drug conjugates of Formula I can be heterogeneous mixtures of such conjugates and antibodies linked to one, two, three, four, or more drug moieties.

[0181] In some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, as in the embodiments provided herein, when the linkage is a cysteine ​​thiol, the antibody may have only one or several cysteine ​​thiol groups or only one or several sufficiently reactive thiol groups through which the linker can be attached. In certain embodiments, higher drug loading (e.g., p>5) may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading of the ADCs of the present 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, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8 and may be about 2 to about 5. See US2005 / 0238649(A1).

[0182] In certain embodiments, fewer than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with, for example, the drug-linker intermediate or linker reagent, as discussed below. In general, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to drug moieties, and in fact, most cysteine ​​thiol residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody may be reduced by a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

[0183] The loading (drug / antibody ratio) of ADCs can be controlled in different ways, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions for cysteine ​​thiol modification.

[0184] It is understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent and subsequently with a drug moiety reagent, the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay that is specific for the antibody and the drug. Individual ADC molecules may be identified in the mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (see, e.g., McDonagh et al (2006) Prot. Engr. 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, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). (See, Proceedings of the AACR Research, 2004 Annual Meeting, March 27-31, 2004, Volume 45, March 2004.) In certain embodiments, homogenous ADCs having a single loading value may be isolated from a complex mixture by electrophoresis or chromatography.

[0185] 5. Exemplary Methods of Preparing Immunoconjugates ADCs of formula I can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including (1) reacting a nucleophilic group of an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with a drug moiety, D, and (2) reacting a nucleophilic group of a drug moiety with a bivalent linker reagent to form DL via a covalent bond, followed by reaction with a nucleophilic group of an antibody. An exemplary method for preparing ADCs of formula I via the latter route is described in US2005 / 0238649(A1), which is expressly incorporated herein by reference.

[0186] Nucleophilic groups on an antibody include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups on which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents, including (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides, (ii) alkyl and benzyl halides, such as haloacetamides, and (iii) aldehyde, ketone, carboxyl, and maleimide groups, to form covalent bonds. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treating them with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the antibody is fully or partially reduced. Thus, each cysteine ​​bridge would theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through modification of lysine residues, for example by reacting the lysine residues with 2-iminothiolane (Traut's reagent), resulting in the conversion of amines to thiols. Reactive thiol groups can also be introduced into antibodies by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a variant antibody containing one or more non-natural cysteine ​​amino acid residues).

[0187] The antibody-drug conjugates of the present disclosure may also be produced by reaction between electrophilic groups on an antibody, such as aldehyde or ketone carbonyl groups, and nucleophilic groups on a linker reagent or on a drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, an antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or on a drug. In another embodiment, the sugars of a glycosylated antibody may be oxidized, for example, with a periodate oxidation reagent, to form aldehyde or ketone groups that can react with amine groups on a linker reagent or drug moiety. The resulting imine Schiff base groups may form stable bonds or may be reduced, for example, with a borohydride reagent, to form stable amine bonds. In one embodiment, the carbohydrate moiety of a glycosylated antibody can be reacted with either galactose oxidase or sodium metaperiodate to yield carbonyl (aldehyde and ketone) groups within the antibody that can react with appropriate groups on a drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate, resulting in the production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146, US5362852). Such aldehydes can react with drug moieties or linker nucleophiles.

[0188] Nucleophilic groups on the drug moiety include, but are not limited to, (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide groups that can react with electrophilic groups on linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups, to form covalent bonds.

[0189] The compounds of the present disclosure expressly contemplate, but are not limited to, ADCs prepared with the following crosslinker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available, e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA (see pages 467-498 of the 2003-2004 Applications Handbook and Catalog).

[0190] Immunoconjugates comprising an antibody and a cytotoxic agent can also be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026.

[0191] B. Surfactants The present disclosure provides compositions comprising an anti-CD79b immunoconjugate and a surfactant. In certain embodiments, the surfactant is a polysorbate (e.g., polysorbate 20 and polysorbate 80); a poloxamer (e.g., poloxamer 188); N-octyl-β-D glucopyranoside (OG); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; lauroamidopropyl betaine, myristyl betaine, or cetyl betaine; lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol, ethylene glycol-propylene glycol copolymers (e.g., Pluronics or PF68), or any combination thereof.

[0192] In some embodiments, the surfactant herein is non-ionic. In certain 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 particular embodiment, the surfactant is PS20. In another embodiment, the surfactant herein is PS80.

[0193] In certain embodiments, the surfactant (eg, PS20) in the pharmaceutical composition is at a concentration of at least about 0.01% by weight / volume (ie, 0.1 mg / ml) and about 0.20% by weight / volume (ie, 2.0 mg / ml). In certain embodiments, the surfactant (e.g., PS20) in the pharmaceutical composition is at least about 0.01% by weight (i.e., 0.1 mg / ml), at least about 0.02% by weight (i.e., 0.2 mg / ml), at least about 0.03% by weight (i.e., 0.3 mg / ml), at least about 0.04% by weight (i.e., 0.4 mg / ml), at least about 0.05% by weight (i.e., 0.5 mg / ml), at least about 0.06% by weight (i.e., 0.6 mg / ml), at least about 0.07% by weight (i.e., 0.7 mg / ml), at least about 0.08% by weight (i.e., 0.8 mg / ml), at least about 0.09% by weight (i.e., 0.9 mg / ml), at least about 0.10% by weight (i.e., 1 mg / ml), at least about 0.15% by weight (i.e., 1 mg / ml), at least about 0.16% by weight (i.e., 1 mg / ml), at least about 0.18% by weight (i.e., 1 mg / ml), at least about 0.19% by weight (i.e., 1 mg / ml), at least about 0.20% by weight (i.e., 1 mg / ml), at least about 0.21% by weight (i.e., 1 mg / ml), at least about 0.22% by weight (i.e., 1 mg / ml), at least about 0.23% by weight (i.e., 1 mg / ml), at least about 0.24% by weight (i.e., 1 mg / ml), at least about 0.25% by weight (i.e., 1 mg / ml), at least about 0.26% by weight ( % by weight (i.e., 1.7 mg / ml), at least about 0.15% by weight (i.e., 1.5 mg / ml), at least about 0.16% by weight (i.e., 1.6 mg / ml), at least about 0.17% by weight (i.e., 1.7 mg / ml), at least about 0.18% by weight (i.e., 1.8 mg / ml), at least about 0.19% by weight (i.e., 1.9 mg / ml), or at least about 0.20% by weight (i.e., 2.0 mg / ml) (including any ranges therebetween). In one particular embodiment, the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of at least about 0.06% by weight or volume (i.e., 0.6 mg / ml). In another particular embodiment, the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of at least about 0.12% by weight or volume (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 stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at 30° C., or for at least about any of 24, 48, or 72 hours when stored at 2° C. to 8° C.

[0194] In certain embodiments, the surfactant (eg, PS20) in the pharmaceutical composition is at a concentration of at least about 0.01% by weight / volume (ie, 0.1 mg / ml) and about 0.20% by weight / volume (ie, 2.0 mg / ml). In certain embodiments, the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at a concentration of at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.10%, at least 0.11%, at least 0.12%, at least 0.13%, at least 0.14%, at least 0.15%, at least 0.16%, at least 0.17%, at least 0.18%, at least 0.19%, or at least 0.20% by weight (including any ranges therebetween). In one particular embodiment, the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at a concentration of at least 0.06% by weight / volume. In another particular embodiment, the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at a concentration of at least 0.12% by weight / volume. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized cake. In some embodiments, the lyophilized pharmaceutical composition is contained in a glass vial.

[0195] In certain embodiments, the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at a concentration of at least about 0.1 to about 2.0 mg / ml. In certain embodiments, the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at a concentration of 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 (including any range between these values). In one particular embodiment, the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at a concentration of at least about 0.6% mg / ml. In another particular embodiment, the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at a concentration of at least 1.2 mg / ml. In some embodiments, the liquid pharmaceutical composition is reconstituted (e.g., reconstituted using SWFI) from a lyophilized pharmaceutical composition (such as a lyophilized cake). In some embodiments, the liquid pharmaceutical composition is contained in a glass vial. In some embodiments, the liquid pharmaceutical composition is stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at 30°C, or for at least about any of 24, 48, or 72 hours when stored at 2°C to 8°C.

[0196] In one exemplary embodiment, the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at a concentration of 0.06% by weight / volume. In another exemplary embodiment, the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at a concentration of 0.12% by weight / volume. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized cake. In some embodiments, the lyophilized pharmaceutical composition is contained within a glass vial.

[0197] In one exemplary embodiment, the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at a concentration of 0.6 mg / ml. In another exemplary embodiment, the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at a concentration of 1.2 mg / ml. In some embodiments, the liquid pharmaceutical composition is reconstituted from a lyophilized composition (such as a lyophilized cake) (e.g., reconstituted using SWFI). In some embodiments, the liquid pharmaceutical composition is contained in a glass vial. In some embodiments, the liquid pharmaceutical composition is stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at 30° C., or for at least about any of 24, 48, or 72 hours when stored at 2° C. to 8° C. In some embodiments, the liquid pharmaceutical composition is stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at 30° C., or for at least about any of 24, 48, or 72 hours when stored at 2° C. to 8° C.

[0198] In some embodiments, the anti-CD79b immune complex in the pharmaceutical composition is at a concentration of 10 mg / ml and the surfactant in the pharmaceutical composition is at a concentration of at least 0.06% by weight (i.e., 0.6 mg / ml). In some embodiments, the anti-CD79b immune complex in the pharmaceutical composition is at a concentration of 10 mg / ml and the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of at least 0.12% by weight (i.e., 1.2 mg / ml). In some embodiments, the pharmaceutical composition is a reconstituted composition (e.g., reconstituted using SWFI from a lyophilized composition). In some embodiments, the pharmaceutical composition is contained within a glass vial. In some embodiments, the liquid pharmaceutical composition is stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours when stored at 30° C., or for at least about any of 24, 48, or 72 hours when stored at 2° C. to 8° C.

[0199] In some embodiments, the anti-CD79b immune complex in the pharmaceutical composition is at a concentration of 10 mg / ml and the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of 0.06% by weight or volume (i.e., 0.6 mg / ml). In some embodiments, the anti-CD79b immune complex in the pharmaceutical composition is at a concentration of 10 mg / ml and the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of 0.12% by weight or volume (i.e., 1.2 mg / ml). In some embodiments, the pharmaceutical composition is a reconstituted composition (e.g., reconstituted using SWFI from a lyophilized composition). In some embodiments, the pharmaceutical composition is contained within a glass vial.

[0200] In some embodiments, the anti-CD79b immune complex in the pharmaceutical composition is at a concentration of 20 mg / ml and the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of at least 0.06% by weight or volume (i.e., 0.6 mg / ml). In some embodiments, the anti-CD79b immune complex in the pharmaceutical composition is at a concentration of 20 mg / ml and the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of at least 0.12% by weight or volume (i.e., 1.2 mg / ml). In some embodiments, the pharmaceutical composition is a reconstituted composition (e.g., reconstituted using SWFI from a lyophilized composition). In some embodiments, the pharmaceutical composition is contained within a glass vial.

[0201] In some embodiments, the anti-CD79b immunoconjugate in the pharmaceutical composition is at a concentration of 20 mg / ml and the surfactant (e.g., PS20) in the pharmaceutical composition is at a concentration of 0.06% by weight or volume (i.e., 0.6 mg / ml). In some embodiments, the anti-CD79b immunoconjugate in the pharmaceutical composition is at a concentration of 20 mg / ml and the surfactant in the pharmaceutical composition is at a concentration of 0.12% by weight or volume (i.e., 1.2 mg / ml). In some embodiments, the liquid pharmaceutical composition is stable for at least about any of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours when stored at 30° C., or for at least about any of 24, 48, or 72 hours when stored at 2° C. to 8° C.

[0202] In some embodiments, the pharmaceutical composition is a liquid composition suitable for intravenous administration. In some embodiments, the liquid composition suitable for administration comprises about 0.0432 and about 0.162 mg / ml of a surfactant (e.g., polysorbate 20). In some embodiments, the liquid composition comprises about 0.72 mg / ml to about 2.7 mg / ml of polatuzumab vedotin. In some embodiments, the liquid composition suitable for intravenous administration comprises about 0.72 mg / ml of polatuzumab vedotin and about 0.0432 mg / ml of polysorbate 20. In some embodiments, the liquid composition suitable for intravenous administration comprises about 2.7 mg / ml of polatuzumab vedotin and about 0.162 mg / ml of polysorbate 20.

[0203] C. Buffer According to the present disclosure, a pharmaceutical composition comprising a buffering agent is provided. Without wishing to be bound by theory, the use of a buffer maintains the pH of the pharmaceutical composition during the preparation, storage, and use of the composition. Non-limiting examples of buffering agents include histine, succinic acid, 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)propan-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)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), or phosphate buffered saline (PBS). Other suitable buffers can be acetic acid in salt, citric acid in salt, boric acid in salt, and phosphoric acid in salt.

[0204] In exemplary embodiments of the present disclosure, the buffer is a histidine buffer or a succinate buffer. In one particular embodiment, the succinate buffer is a sodium succinate buffer.

[0205] In some embodiments, the buffer is at a concentration of about 10 mM to about 200 mM. In a particular embodiment, the buffer is at a concentration of about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, about 100 mM, about 101 mM, about 102 mM, about 103 mM, about 104 mM, about 105 mM, about 106 mM, about 107 mM, about 108 mM, about 109 mM, about 110 mM, about 120 mM, about 121 mM, about 122 mM, about 130 mM, about 131 mM, about 132 mM, about 133 mM, about 134 mM, about 135 mM, about 136 mM, about 13 The concentration is about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM.

[0206] In another specific embodiment, 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, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 151 mM, 160 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, 105 mM, 106 mM, 107 mM, 108 mM, 110 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, The concentration is about 190 mM, 195 mM, or 200 mM.

[0207] In an exemplary embodiment of the present disclosure, the sodium succinate buffer has a concentration of about 10 mM to about 200 mM. In one embodiment, the sodium succinate buffer has a concentration of about 10 mM. In another embodiment, the sodium succinate buffer has a concentration of 10 mM.

[0208] In some embodiments, the compositions of the present disclosure have a pH of about 5.0 to about 6.0. In certain embodiments, the buffer 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 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.

[0209] In one exemplary embodiment, the composition of the present disclosure has a pH of about 5.3. In another exemplary embodiment, the composition of the present disclosure has a pH of 5.3.

[0210] D.sugar According to the present disclosure, a pharmaceutical composition is provided that includes a sugar. Without wishing to be bound by theory, the sugar functions to promote cryoprotection of the pharmaceutical composition, thereby preventing aggregation and maintaining the chemical and physical stability of the pharmaceutical composition. In certain embodiments, the sugar is glucose, fructose, sucrose, trehalose, arginine, glycerin, proline, dextran, dextran 40, glycerol, mannitol, or sorbitol.

[0211] In some embodiments, the sugar is selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, dextran 40, and trehalose. In an exemplary embodiment, the sugar is sucrose.

[0212] In some embodiments, the sugar is at a concentration of about 50 mM to about 300 mM. In a particular embodiment, the buffer is at a concentration of 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 is at a concentration of about 100 mM to about 260 mM.

[0213] In some embodiments, the sugar is at a concentration of 50 mM to 300 mM. In a particular embodiment, the buffer is at a concentration of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 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 is at a concentration of 100 mM to 260 mM.

[0214] In one exemplary embodiment of the present disclosure, the sucrose is at a concentration of about 120 mM. In another exemplary embodiment, the sucrose is at a concentration of 120 mM.

[0215] In another aspect, the disclosure provides a pharmaceutical composition produced by lyophilization of a liquid composition comprising 20 mg / ml anti-CD79b immunoconjugate, 0.12% weight / volume polysorbate 20, and 120 mM sucrose in 10 mM sodium succinate buffer, wherein the liquid composition has a pH of 5.3, and the anti-CD79b immunoconjugate has the formula: TIFF2025032097000018.tif25170, wherein The pharmaceutical composition is provided wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p is a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5).

[0216] In some embodiments of the disclosure, an anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 9 and the light chain comprises the amino acid sequence of SEQ ID NO: 10.

[0217] E. Stability In some embodiments, the physical stability, chemical stability, or biological activity of the pharmaceutical composition in liquid state or lyophilized form is evaluated or measured. Any method known in the art and described in the Examples herein can be used to evaluate the stability and biological activity of the pharmaceutical composition of the present disclosure. For example, the stability of the antibody in the pharmaceutical composition can be measured by, but not limited to, size exclusion chromatography (SEC or SE-HPLC), imaging capillary isoelectric focusing (icIEF), peptide mapping, low volume light obscuration (HIAC) assay, and capillary electrophoresis (CE) techniques (such as CE-sodium dodecyl sulfate (CE-SDS) and CE-glycan analysis). In some embodiments, a stable pharmaceutical composition (or formulation) is a formulation in which the immune complex therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage, for example, upon storage for a particular time or period (hours, days, months, years, etc.) under certain conditions (temperature, relative humidity, residual moisture, presence or absence of light, after a period of agitation, etc.). In some embodiments, an immunoconjugate present in the pharmaceutical composition is biologically stable (e.g., retains its biological activity) if the biological activity of the immunoconjugate (and / or the antibody portion of the immunoconjugate, such as an anti-CD79b antibody) at a given time is within the range accepted by the national or regional regulatory authority for pharmaceutical products (e.g., the Federal Drug Administration (FDA) in the United States, the Therapeutic Goods Administration (TGA) in Australia, the European Medicines Agency (EMA) in the European Union, etc.). For example, the biological activity of an immunoconjugate (such as an immunoconjugate comprising an anti-CD79b antibody) or a pharmaceutical composition comprising such an immunoconjugate can be measured by its ability to bind to an antigen (such as the ability of an anti-CD79b antibody to bind to CD79b, e.g., human CD79b). For example, antibody binding affinity measurements can be measured using a number of assays known in the art, 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 comprising an anti-CD79b antibody) is measured, for example, by its ability to inhibit cell growth and / or cell proliferation, or to induce cell death, including programmed cell death (apoptosis), for example, in vitro or in vivo. Further details regarding assays for measuring the biological activity of anti-CD79b antibodies or immunoconjugates comprising anti-CD79b antibodies are provided in U.S. Patent No. 8,088,387, the contents of which are incorporated herein by reference in their entirety.

[0218] In some embodiments, the pharmaceutical compositions of the present disclosure are 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 when protected from light.

[0219] In some embodiments, the pharmaceutical compositions of the present disclosure are stable at 2° C.-8° C. (e.g., 5° C.±3° 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 when protected from light.

[0220] In some embodiments, a pharmaceutical composition of the present disclosure is stable at 25° C.-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.

[0221] In one exemplary embodiment, the composition has a stability for at least about 48 months when protected from light at 5° C.±3° C. In another exemplary embodiment, the composition has a stability for about 48 months when protected from light at 5° C.±3° C.

[0222] In certain embodiments, the lyophilized form (e.g., lyophilized cake) of the pharmaceutical composition of the present disclosure may be tested for stability. In some embodiments, under storage conditions (5°C±3°C and protected from light), the lyophilized cake does not demonstrate any significant changes in structure, color, appearance, or moisture content. In one particular embodiment, the lyophilized cake has a moisture content of less than 5% under storage conditions. In another particular embodiment, the lyophilized cake is smooth and not nicked under storage conditions. In certain embodiments of the present disclosure, the lyophilized cake is stable under storage conditions for at least about 48 months.

[0223] In some embodiments, the stability of the pharmaceutical composition of the present disclosure is measured by size exclusion chromatography high performance (SE-HPLC). In an exemplary embodiment, the composition has a main peak area % of at least 95.0 as measured by SE-HPLC.

[0224] In some embodiments, the lyophilized pharmaceutical compositions provided herein (e.g., cakes) are stable for at least about 44 months (e.g., any of about 30, 35, 40, 45, 50, or 55 months) when stored at a temperature of about 2° C. to about 8° C. over a residual moisture content range of about 0.3% to about 3.2% by weight (including any ranges between these values). In some embodiments, the lyophilized pharmaceutical compositions provided herein (e.g., cakes) are stable for at least about 7 months (e.g., any of about 2, 3, 4, 5, 6, 7, 8, 8, or 10 months) when stored at about 25° C. over a residual moisture content range of about 0.3% to about 3.2% by weight (including any ranges between these values).

[0225] In some embodiments, the lyophilized pharmaceutical compositions (e.g., cakes) provided herein are robust over (a) protein concentrations of about 17 to about 23 mg / ml, (b) succinic acid concentrations of about 7 to about 23 mM, (c) sucrose concentrations of about 90 to about 150 mM, (d) polysorbate 20 concentrations of about 0.9 to about 1.5 mg / ml, and (e) a pH of about 4.95 to about 5.65. In some embodiments, the lyophilized pharmaceutical compositions (e.g., cakes) comprising any one or more of (a) to (e) above are stable at a temperature of about 2° C. to about 8° C. for up to any of at least about 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.

[0226] G. Compositions for Intravenous Administration The present disclosure provides pharmaceutical compositions and liquid compositions suitable for intravenous (IV) administration. The compositions used for in vivo IV administration should be sterile. This is easily achieved by filtration through a sterile filtration membrane before or after preparation of the pharmaceutical composition. In certain embodiments, the pharmaceutical compositions of the present 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) administration bag.

[0227] In one aspect, the disclosure provides a pharmaceutical composition comprising an anti-CD79b immune conjugate, 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 immune conjugate at a concentration of about 10 mg / ml to about 20 mg / ml, a surfactant at a concentration of at least 0.06% weight by volume (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 the anti-CD79b immune conjugate is represented by the formula: TIFF2025032097000019.tif25170, wherein The pharmaceutical composition is provided, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p has a value of about 1 to about 8 (such as about 2 to about 5, for example, about 3.5).

[0228] In some embodiments, the pharmaceutical composition is reconstituted (1:1 mass:volume) in SWFI. In some embodiments, when reconstituted in water, the pharmaceutical composition forms a liquid pharmaceutical composition comprising 20 mg / ml anti-CD79b immunoconjugate, 0.12% polysorbate 20 by weight, and 120 mM sucrose in 10 mM sodium succinate buffer, wherein the liquid pharmaceutical composition has a pH of 5.3.

[0229] In some embodiments, the pharmaceutical composition is reconstituted in SWFI and then diluted into an isotonic buffer in an IV administration bag, and the surfactant concentration upon dilution into the IV administration bag is at least 0.003% by weight / volume. In one embodiment, the surfactant concentration upon dilution into the IV administration bag is at least 0.004% by weight / volume. In an exemplary embodiment, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one particular embodiment, the sucrose is at a concentration of 120 mM. In some embodiments, the succinate buffer is a sodium succinate buffer. In one particular exemplary embodiment, the sodium succinate buffer is at a concentration of 10 mM.

[0230] In some embodiments, the stability of the pharmaceutical compositions of the present disclosure when reconstituted in water is measured by aggregation, pH, turbidity, number of visible and / or subvisible particles, or amount of free (e.g., uncomplexed) drug. In certain embodiments, UV spectrophotometry, SE-HPLC, hydrophobic interaction chromatography (HIC), icIEF, or HIAC, or a combination thereof, can be used to assess stability. In some embodiments, the pharmaceutical compositions when reconstituted in water have a stability of up to about 1 day, up to about 2 days, or up to about 3 days at 30° C. In some embodiments, the pharmaceutical compositions when reconstituted in water have a stability of up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, or up to about 7 days at 5° C.±3° C.

[0231] In certain embodiments, the stability of the composition reconstituted in water is measured by size-exclusion high performance liquid chromatography (SE-HPLC). In an exemplary embodiment, the composition has a main peak area % of at least 95.0 as measured by SE-HPLC.

[0232] In certain embodiments, the composition has a HMWS (%) of less than 4.0 as measured by SE-HPLC. In certain embodiments, the composition has a LMWS (%) of less than 1.0 as measured by SE-HPLC. In some embodiments, the composition has a main peak (area %) of at least 95.0 and a HMWS of less than 4.0 as measured by SE-HPLC. In some embodiments, the composition has a main peak (area %) of at least 95.0, a HMWS (%) of less than 4.0, and a LMWS (%) of less than 1.0 as measured by SE-HPLC.

[0233] In certain embodiments, the stability of the reconstituted composition in water is measured by imaging capillary isoelectric focusing (icIEF).In one embodiment, the composition has a main peak (area%) of at least 73.0, an acidic area (area%) of at most 25.5, and a basic area (area%) of at most 10.0, as measured by icIEF.In an exemplary embodiment, the composition has a main peak (area%) of at least 58.0, an acidic area (area%) of at most 32.0, and a basic area (area%) of at most 12.0, as measured by icIEF.

[0234] In some embodiments, a reconstituted pharmaceutical composition comprising 20 mg / mL polatuzumab vedotin in 10 mM succinic acid, 120 mM sucrose, 1.2 mg / mL polysorbate 20, pH 5.3, is physicochemically stable after 72 hours of storage at a temperature of about 2° C. to about 8° C. In some embodiments, storage of the reconstituted pharmaceutical composition for 72 hours at a temperature of about 2° C. to about 8° C. does not decrease the biological activity of polatuzumab vedotin. In some embodiments, a reconstituted pharmaceutical composition comprising 20 mg / mL polatuzumab vedotin in 10 mM succinic acid, 120 mM sucrose, 1.2 mg / mL polysorbate 20, pH 5.3, is physicochemically stable after 24 hours of storage at 30° C. with exposure to ambient light. In some embodiments, storage of the reconstituted pharmaceutical composition at 30° C. for 24 hours with exposure to ambient light does not decrease the biological activity of polatuzumab vedotin.

[0235] In one aspect, the disclosure provides a liquid composition comprising an anti-CD79b immune complex, a surfactant, a succinate buffer, and a sugar, wherein the anti-CD79b immune complex is at a concentration of about 10 mg / ml to about 20 mg / ml, the surfactant is at a concentration of at least 0.06% weight / volume (i.e., 0.6 mg / ml), the succinate buffer is at a concentration of about 10 mM to about 200 mM, and the sugar is at a concentration of about 100 mM to about 260 mM, the liquid composition has a pH of 5.3, and the anti-CD79b immune complex is represented by the formula: TIFF2025032097000020.tif25170, wherein The liquid composition is provided, wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the light chain comprises (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain comprises (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, and p has a value of about 1 to about 8 (e.g., about 2 to about 5, such as about 3.5).

[0236] In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In one exemplary embodiment, the sucrose is at a concentration of 120 mM. In some embodiments, the succinate buffer is a sodium succinate buffer. In one particular exemplary embodiment, the sodium succinate buffer is at a concentration of 10 mM.

[0237] In certain embodiments, the liquid composition of the present disclosure is dissolved in a buffer solution, which may be, but is not limited to, normal saline (0.9% by weight sodium chloride), half saline (0.45% by weight sodium chloride), 5% by weight dextrose, lactated Ringer's solution, or a combination thereof.

[0238] In some embodiments, the liquid composition of the present disclosure is dissolved in an isotonic buffer solution.In certain embodiments, the isotonic buffer solution can be freshly prepared or can be contained in a pre-filled IV administration bag.In certain embodiments, the isotonic buffer solution is normal saline (0.9% sodium chloride).In an exemplary embodiment, the liquid composition of the present disclosure dissolved in an isotonic buffer solution is in an IV administration bag.

[0239] In some embodiments, the liquid composition of the present disclosure dissolved in an isotonic buffer in an IV administration bag has a pH of about 4.8 to about 5.8. In a particular 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 the present disclosure dissolved in an isotonic buffer in an IV administration bag has a pH of about 5.3.

[0240] In some embodiments, the stability of the liquid composition in the IV administration bag of the present disclosure is measured by aggregation, pH, turbidity, number of visible and / or subvisible particles, or amount of free (e.g., uncomplexed) drug. In certain embodiments, UV spectrophotometry, SE-HPLC, hydrophobic interaction chromatography (HIC), icIEF, or HIAC, or a combination thereof, can be used to assess the stability of the liquid composition in the IV administration bag.

[0241] In certain embodiments, the liquid composition of the present disclosure in the IV administration bag is stable under static conditions. In certain embodiments, the liquid composition of the present disclosure in the IV administration bag is stable under stirring conditions. In some embodiments, the liquid composition of the present disclosure dissolved in the isotonic buffer solution in the IV administration bag has a stability of up to about 1 hour, up to about 2 hours, up to about 3 hours, up to about 4 hours, up to about 5 hours, up to about 6 hours, up to about 7 hours, or up to about 8 hours at 30°C. In some embodiments, the liquid composition of the present disclosure dissolved in the isotonic buffer solution in the IV administration bag has a stability of up to about 8 hours, up to about 9 hours, up to about 10 hours, up to about 12 hours, up to about 14 hours, up to about 16 hours, up to about 18 hours, up to about 20 hours, up to about 22 hours, or up to about 24 hours at 25°C. In some embodiments, the liquid composition of the present disclosure dissolved in an isotonic buffer in an IV administration bag has a stability of up to about 24 hours, up to about 30 hours, up to about 36 hours, up to about 42 hours, up to about 48 hours, up to about 54 hours, up to about 60 hours, up to about 66 hours, or up to about 72 hours at 5° C.±3° C. In certain embodiments, the isotonic buffer is normal saline.

[0242] In some embodiments, the reconstituted or diluted solutions for injection described herein (e.g., diluted solutions for injection containing an immunoconjugate (e.g., polatuzumab vedotin)) are sterile.

[0243] III. Treatment method It is contemplated that the pharmaceutical or liquid composition of the present disclosure can be used to treat various proliferative disorders, such as those characterized by overexpression of tumor antigens.Exemplary proliferative disorders are cancers.Examples of cancers include, but are not limited to, hematopoietic or blood-related cancers, such as lymphoma, leukemia, myeloma, or lymphatic tumors, as well as spleen and lymph node cancers, and carcinomas, blastomas, and sarcomas. More specific examples of cancers include, for example, high-, intermediate-, and low-grade lymphomas (including B-cell lymphomas such as mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin's lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin's lymphoma, as well as T-cell lymphomas), and B-cell proliferative disorders including leukemias (including secondary leukemias, chronic lymphocytic leukemia (CLL) (such as B-cell leukemia (CD5+ B-lymphocytes)), myeloid leukemias (such as acute myeloid leukemia, chronic myeloid leukemia), lymphocytic leukemias (such as acute lymphoblastic leukemia (ALL)), and myelodysplasia), as well as other hematological and / or B- or T-cell related cancers. Also included are cancers of additional hematopoietic cells, including polymorphonuclear leukocytes, such as basophils, eosinophils, neutrophils, and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells. In certain embodiments, the proliferative disorder of the present disclosure is a recurrent or refractory cancer.

[0244] In certain embodiments, the pharmaceutical or liquid compositions of the present disclosure can be used to treat relapsed leukemia or relapsed lymphoma. In other certain embodiments, the pharmaceutical or liquid compositions of the present disclosure can be used to treat refractory leukemia or refractory lymphoma.

[0245] In one aspect, the disclosure provides a method of treating a proliferative disorder in a patient in need of such treatment, comprising administering to the patient a pharmaceutical or liquid composition described herein.

[0246] In some embodiments, the cancer to be treated is a B cell proliferative disorder. In certain embodiments, the B cell proliferative disorder is selected from the group consisting of lymphoma, myeloma, non-Hodgkin's 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 lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0247] In one embodiment of the present disclosure, the B cell proliferative disorder is non-Hodgkin's lymphoma (NHL). In an exemplary embodiment, the B cell proliferative disorder is diffuse large B cell lymphoma (DLBCL). In a 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).

[0248] 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.

[0249] Cancer may contain CD79b expressing cells such that the anti-CD79b immunoconjugates of the present disclosure can bind to the cancer cells. To determine the expression of CD79b in cancer, various diagnostic / prognostic assays are available. In one embodiment, overexpression of CD79b can be analyzed by IHC. Paraffin-embedded tissue sections from tumor biopsy tissue can be subjected to IHC assay and matched to CD79b protein staining intensity criteria with respect to the degree of staining and in the percentage of tumor cells examined.

[0250] For the prevention or treatment of a disorder, the appropriate dosage of the ADC will depend on the type of disease being treated, the severity and course of the disease, whether the molecule is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician, as described above. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) of the molecule is an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion. Typical daily dosages can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. Exemplary dosages of the ADC administered to a patient are in the range of about 0.1 to about 10 mg per kg of the patient's body weight. For repeated administration over several days or more depending on the condition, treatment is sustained until a desired suppression of disease symptoms occurs.

[0251] To treat or prevent a proliferative disorder, the pharmaceutical or liquid composition of the present disclosure is administered via intravenous infusion. The dosage administered via infusion is about 1 μg / m per dose. 2 ~Approx. 10,000μg / m 2 , typically one dose per week for a total of 1, 2, 3, or 4 doses. Alternatively, the dosage range is about 1 μg / m 2 ~Approx. 1000μg / m 2 , about 1μg / m 2 ~about 800μg / m 2 , about 1μg / m 2~about 600μg / m 2 , about 1μg / m 2 ~about 400μg / m 2 , about 10μg / m 2 ~about 500μg / m 2 , about 10μg / m 2 ~about 300μg / m 2 , about 10μg / m 2 ~about 200μg / m 2 , and about 1 μg / m 2 ~about 200μg / m 2 The dose may be administered once a day, once a week, multiple times a week but less than once a day, multiple times a month but less than once a day, multiple times a month but less than once a week, once a month, or intermittently to alleviate or relieve symptoms of the disease. Administration may continue at any of the intervals disclosed until remission of the tumor or symptoms of the lymphoma, leukemia being treated. Administration may continue after remission or relief of symptoms is achieved, if such remission or relief is prolonged by such continued administration.

[0252] To determine the expression of CD79b in cancer, various detection assays are available. In one embodiment, the overexpression of CD79b polypeptide can be analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsy tissue can be subjected to IHC assay and matched with the CD79b protein staining intensity criteria as follows: Score 0: no staining or membranous staining is observed in less than 10% of the tumor cells. Score 1+: A faint / barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in a part of their membrane. Score 2+: weak to moderate complete membrane staining is observed in more than 10% of the tumor cells. Score 3+: A moderate to intense complete membrane staining is observed in more than 10% of the tumor cells.

[0253] Tumors with a score of 0 or 1+ for expression of CD79b polypeptide can be characterized as not overexpressing CD79b, while tumors with a score of 2+ or 3+ can be characterized as overexpressing CD79b.

[0254] Alternatively, or in addition, a FISH assay, such as INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois), can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the degree of CD79b overexpression in the tumor, if present.

[0255] CD79b overexpression or amplification can be assessed using an in vivo detection assay, for example, by administering a molecule (such as an antibody) that binds to the molecule to be detected and is tagged with a detectable label (e.g., a radioisotope or fluorescent label) and externally scanning the patient for localization of the label.

[0256] Combination therapy Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy, and chemotherapy. Anti-CD79b immunoconjugate therapy may be particularly desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well, as well as in metastatic disease where radiation therapy has limited utility. The tumor-targeted anti-CD79b immunoconjugates of the present disclosure are useful for palliating CD79b-expressing cancers at the time of initial diagnosis or during recurrence of the disease. For therapeutic use, anti-CD79b immunoconjugates may be used alone or in combination therapy with, for example, hormones, antiangiogenic agents, or radiolabeled compounds, or surgery, cryotherapy, and / or radiation therapy. Administration of the pharmaceutical or liquid compositions of the present disclosure can be performed in combination with other forms of conventional therapy, either consecutively, prior to, or subsequent to conventional therapy. Chemotherapeutic agents such as TAXOTERE® (docetaxel), TAXOL® (paclitaxel), estramustine, and mitoxantrone are used in treating cancer, particularly in low-risk patients. In the methods of the present disclosure for treating or alleviating cancer, the cancer patient may be administered an anti-CD79b antibody in combination with treatment with one or more of the aforementioned chemotherapeutic agents. In particular, combination therapy with paclitaxel and modified derivatives (see, e.g., EP0600517) is contemplated. The pharmaceutical or liquid composition of the present disclosure may be administered together with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, the pharmaceutical or liquid composition of the present disclosure is administered in combination with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses the dosages of these agents that have been used in the treatment of various cancers. The dosing regimens and dosages of these above-mentioned chemotherapeutic agents that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease, and other factors known to physicians in the art, and can be determined by a physician.

[0257] In another embodiment, the pharmaceutical or liquid compositions of the present disclosure may be administered in combination with one or more chemotherapeutic agents or growth inhibitory agents (including simultaneous administration of a cocktail of different chemotherapeutic agents), or other cytotoxic agent(s) or other therapeutic agent(s) that also inhibit tumor growth. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea, and hydroxyurea taxanes (such as paclitaxel and docetaxel), and / or anthracycline antibiotics. Preparation and dosing schedules of such chemotherapeutic agents may be used according to the manufacturer's instructions or as empirically determined by one of skill in the art. Preparation and dosing schedules of such chemotherapeutic agents are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The pharmaceutical or liquid composition of the present disclosure can be combined with an anti-hormonal compound, such as an anti-estrogen compound (such as tamoxifen), an anti-progesterone (such as onapristone (see EP616812)), or an anti-androgen (such as flutamide) in dosages known for such molecules. If the cancer being treated is an androgen-independent cancer, the patient may have previously been subjected to anti-androgen therapy, and the pharmaceutical or liquid composition of the present disclosure can be administered to the patient after the cancer has become androgen-independent.

[0258] Sometimes, it may also be beneficial to co-administer a cardioprotectant or one or more cytokines to the patient (to prevent or reduce myocardial dysfunction associated with this therapy). In addition to the above treatment regimens, the patient may be subjected to surgical removal of cancer cells and / or radiation therapy (e.g., external beam radiation, or therapy with a radiolabeled agent such as an antibody) prior to, concurrently with, or following administration of the pharmaceutical or liquid composition of the present disclosure. Suitable dosages for any of the above co-administered agents are those currently in use and may be reduced due to the combined action (synergy) of the agent with other anti-CD79b antibodies.

[0259] In some embodiments, the pharmaceutical or liquid compositions of the present disclosure may be administered to a patient in combination with an effective amount of another therapeutic agent, hi 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 radiation therapy, corticosteroids, cancer vaccines, and growth inhibitory agents. In another embodiment, the therapeutic agent is selected from one or more of tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab, vincristine, imatinib mesylate, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, methotrexate, vinblastine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, bortezomib, melphalan, prednisone, and docetaxel.

[0260] In some embodiments, the therapeutic agent is an anti-CD20 antibody.

[0261] In certain embodiments, the pharmaceutical or liquid composition of the present disclosure may be administered in combination with an effective amount of an anti-CD20 antibody to treat a B cell proliferative disorder. 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 certain embodiments, the anti-CD20 antibody is rituximab.

[0262] In some embodiments, the therapeutic agent is a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent comprises one or more of cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone. In another embodiment, the chemotherapeutic agent comprises cyclophosphamide, doxorubicin, and prednisone.

[0263] In some embodiments, the pharmaceutical or liquid compositions of the present disclosure may be administered in combination with an effective amount of an anti-CD20 antibody and a chemotherapeutic agent, hi one embodiment, the chemotherapeutic agent comprises cyclophosphamide, doxorubicin, and prednisone.

[0264] In some embodiments, the pharmaceutical or liquid composition of the present disclosure may be administered in combination with an effective amount of an anti-CD20 antibody and a chemotherapeutic agent to treat a B cell proliferative disorder. In one embodiment, the B cell proliferative disorder is non-Hodgkin's lymphoma (NHL). In another embodiment, the B cell proliferative disorder is diffuse large B cell lymphoma (DLBCL). In one particular embodiment, the DLBCL is relapsed / refractory DLBCL. In another embodiment, the DLBCL is relapsed NHL or refractory NHL. In another particular embodiment, the proliferative disorder is follicular lymphoma (FL). In one particular embodiment, the anti-CD20 antibody is rituximab.

[0265] In one embodiment, the pharmaceutical or liquid composition of the present disclosure may be used in combination with an anti-CD20 antibody (either a naked antibody or an 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 composition of the present disclosure may be used in combination with an anti-VEGF antibody (e.g., Avastin®).

[0266] The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. The combination administration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, preferably with a period during which both (or all) active agents simultaneously exert their biological activity.

[0267] Suitable dosages for any of the above co-administered agents are those currently in use and may be lowered due to the combined action (synergy) of newly identified agents and other chemotherapeutic agents or treatments.

[0268] Combination therapy can provide "synergistic effects" and be found to be "synergistic", i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects resulting from the use of the compounds separately. Synergistic effects can be obtained when the active ingredients are (1) co-formulated and administered or co-delivered in a combined unit dosage formulation, (2) delivered in alternation or parallel as separate formulations, or (3) by some other regimen. When delivered in alternation therapy, synergistic effects can be obtained when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation therapy, the effective dosage of each active ingredient is administered sequentially, i.e., stepwise, whereas in combination therapy, the effective dosages of two or more active ingredients are administered together.

[0269] IV. Manufactured Articles and Kits Another embodiment of the present disclosure is an article of manufacture containing materials useful for treating, preventing, and / or diagnosing a proliferative disorder. In one embodiment, the article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. These containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing, and / or diagnosing a cancer condition and may have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-CD79b immunoconjugate of the present disclosure. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further include instructions for administering the composition to a cancer patient. In addition, the article of manufacture may further include a second container containing a pharma- ceutically acceptable buffer, such as sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0270] In some embodiments, the article of manufacture is a container such as a bottle, vial, syringe, etc., that contains the pharmaceutical composition of the present disclosure. In some embodiments, the article of manufacture is a container such as a bottle, vial, syringe, etc., that contains a lyophilized pharmaceutical composition of the present disclosure (such as a cake). In some embodiments, the article of manufacture is a container such as a bottle, vial, syringe, etc., that contains a reconstituted lyophilized pharmaceutical composition of the present disclosure. In one particular embodiment, the article of manufacture is a container that contains a lyophilized pharmaceutical composition that has been reconstituted with sterile water for injection (SWFI).

[0271] In some embodiments, the article of manufacture is a plastic container containing a pharmaceutical composition of the present disclosure. In some embodiments, the article of manufacture is a glass container containing a pharmaceutical composition of the present disclosure.

[0272] In some embodiments, the article of manufacture is a glass vial containing the pharmaceutical composition of the present disclosure. In one particular embodiment, the article of manufacture is a glass vial containing the lyophilized pharmaceutical composition of the present disclosure (such as a cake). In one particular embodiment, the article of manufacture is a glass vial containing the lyophilized pharmaceutical composition of the present disclosure (such as a cake) that has been reconstituted. In one particular embodiment, the article of manufacture is a glass vial containing the lyophilized pharmaceutical composition of the present disclosure (such as a cake) that has been reconstituted with sterile water for injection (SWFI). In certain embodiments, the product is an IV administration bag containing the pharmaceutical composition or liquid composition of the present disclosure.The IV administration bag can be made 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 administration bag is a polyolefin (PO) bag, a polypropylene (PP) bag, a polyethylene (PE) bag, or a polyvinyl chloride (PVC) bag.

[0273] In some embodiments, an article of manufacture containing a pharmaceutical or liquid composition of the disclosure has a volume of at least about 5 milliliters (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 150 mL, at least about 105 mL, at least about 150 mL, at least about 155 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, at least about 180 mL, at least about 185 mL, at least about 190 mL, at least about 200 mL, at least about 205 mL, at least about 210 mL, at least about 220 mL, at least about 230 mL, at least about 240 mL, at least about 250 mL, at least about 260 mL, at least about 270 mL, at least about 280 mL, at least about 290 mL, at least about 300 mL, at least about 310 mL, at least about 320 mL, at least about 330 mL, at least about 340 mL, at least about 350 mL, at least about 350 mL, at least about 360 mL, at least about 370 mL, at least about 380 mL, at least about 390 mL, at least about 400 mL, at least The bag may contain a volume of 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, 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 of manufacture is a PO bag that can contain a volume of at least about 25 mL, at least about 50 mL, or at least about 100 mL. In some embodiments, the article of manufacture is a PVC bag that can contain a volume of at least about 25 mL, at least about 50 mL, or at least about 100 mL.

[0274] In certain embodiments, the article of manufacture is an IV administration bag containing the pharmaceutical or liquid composition of the present disclosure and a buffer solution. In a particular embodiment, the buffer solution can be normal saline, half saline, 5% by weight dextrose, lactated Ringer's solution, or a combination thereof. In one embodiment, the article of manufacture is an IV administration bag containing the pharmaceutical or liquid composition of the present disclosure dissolved in half saline.

[0275] In some embodiments, the article of manufacture is an IV administration bag comprising a pharmaceutical or liquid composition of the present disclosure and an isotonic buffer, hi one embodiment, the article of manufacture is an IV administration bag comprising a pharmaceutical or liquid composition of the present disclosure dissolved in normal saline.

[0276] In one exemplary embodiment, the article of manufacture is a PVC bag containing a pharmaceutical or liquid composition of the present disclosure and normal saline, capable of containing a volume of at least about 100 mL. In another exemplary embodiment, the article of manufacture is a PVC bag containing a pharmaceutical or liquid composition of the present disclosure and normal saline, capable of containing a volume of about 100 mL.

[0277] In another exemplary embodiment, the article of manufacture is a PO bag comprising a pharmaceutical or liquid composition of the present disclosure and normal saline, capable of containing a volume of at least about 100 mL. In yet another exemplary embodiment, the article of manufacture is a PO bag comprising a pharmaceutical or liquid composition of the present disclosure and semi-saline, capable of containing a volume of at least about 100 mL.

[0278] In any of the embodiments herein, the stable pharmaceutical formulation or liquid pharmaceutical composition can be stored in a container such as a bottle, vial, syringe, or intravenous (IV) administration bag.

[0279] Kits useful for a variety of purposes, such as for CD79b-expressing cell killing assays, are also provided. Similar to an article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-CD79b immunoconjugate of the present disclosure. Additional containers may be included that contain, for example, diluents, detergents, buffers, control antibodies. The label or package insert may provide a description of the composition and instructions for its intended use.

[0280] The following description is provided to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the embodiments described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the embodiments described and shown herein, but are to be accorded the scope consistent with the claims. EXAMPLES

[0281] Example 1: Anti-CD79b immune complex detergent studies The formulation initially used for early phase clinical trials in patients for anti-CD79b-vc-MMAE was a liquid formulation containing histidine acetate buffer with 0.02% polysorbate 20, which was delivered to patients via an intravenous syringe pump. To establish a commercial formulation for the drug product, different concentrations of various non-ionic surfactants were tested to determine whether they could protect and stabilize anti-CD79b-vc-MMAE against aggregation when diluted into an IV dosing bag (which is the route of delivery to patients in a commercial environment). Specifically, the stability of anti-CD79b-vc-MMAE was evaluated both (a) under static conditions in an IV dosing bag, and (b) under air-water interface stress conditions caused by short-term agitation in the IV dosing bag.

[0282] anti-CD79b-vc-MMAE Anti-CD79b-vc-MMAE was diluted in 20 mM histidine acetate, pH 5.5. A 20 mM histidine acetate, pH 5.5 buffer solution was prepared by adding 3.11 g histidine and 1.03 mL glacial acetic acid in 800 mL fresh ultrapure water. The pH was measured to be 5.5 ± 0.1. The resulting solution was adjusted to 1 L using fresh ultrapure water, filtered through a 0.22 μM PES filter, and stored at 2-8 °C. Concentrations of anti-CD79b-vc-MMAE were tested at both (i) 10 mg / mL and (ii) 20 mg / mL by diluting into the filtered buffer to 10 mg / mL or 20 mg / mL, as required. The diluted material was stored at 2-8 °C and protected from light.

[0283] Surfactants The surfactants tested were: (i) Polysorbate 20 (PS20), grade USP, NF, from Croda; (ii) Polysorbate 80 (PS80), grade USP, NF, from Croda; (iii) Poloxamer (P188), grade USP, NF containing 98 ppm butylated hydroxytoluene, from Spectrum Chemicals; and (iv) N-Octyl-β-D Glucopyranoside (OG) C14H28O6, FW: 292.4, from Affymetrix, Anagrade. Preparations of 10%, 1.0%, and 0.5% PS20 stock solutions were prepared as follows: Depending on the target concentration of PS20 in the bag, 10%, 1%, or 0.5% PS20 stock solutions in ultrapure water were used. A 10% PS20 stock solution was prepared by adding 10 g of PS20 into a 100 mL volumetric flask. The flask was filled with 60 mL of ultrapure water and then stirred with a magnetic stirrer to ensure thorough mixing of the solution. After allowing air bubbles to subside for 30 min, the flask was filled to 100 mL with ultrapure water. The solution was transferred to a light-protected container and stored at 2-8 °C for up to 1 week. A similar procedure was used to prepare a 1.0% PS20 solution using 1.0 g of PS20 in 100 mL of ultrapure water. A 1:1 (volume / volume) dilution of the 1.0% PS20 stock was used to make a 0.5% PS20 solution.

[0284] The 0.5% PS80 preparation was as follows: 10% PS20 stock was prepared by adding 10 g of PS80 in a 100 mL volumetric flask. The flask was filled with 50 mL of ultrapure water and then stirred with a magnetic stirrer until the solution was well mixed. The solution was left for 30 minutes to allow air bubbles to subside and then the stirrer was removed. The solution volume was then adjusted to 100 mL using ultrapure water. To obtain the 0.5% PS80 stock solution, 0.5 mL of the 10% PS80 stock solution was adjusted to 10 mL using ultrapure water. The solution was transferred to a light-protected container and stored at 2-8 °C for up to 1 week.

[0285] The 0.5% P188 preparation was as follows: 10% P188 stock was prepared by adding 10 g of P188 in a 100 mL volumetric flask. The flask was filled with 60 mL of ultrapure water and then stirred with a magnetic stirrer until the solution was well mixed. The solution was left for 30 minutes to allow air bubbles to subside and then the stirrer was removed. The solution volume was then adjusted to 100 mL using ultrapure water. To obtain the 0.5% P188 stock solution, 0.5 mL of the 10% P188 stock solution was adjusted to 10 mL using ultrapure water. The solution was transferred to a light-protected container and stored at 2-8 °C for up to 1 week.

[0286] A. Static IV Dosing Bag Study: Anti-CD79b-vc-MMAE was diluted in an IV dosing bag. To mimic the preparation procedure recommended in pharmacy, a volume of saline equal to the total volume of anti-CD79b-vc-MMAE and surfactant added was removed from the IV dosing bag. The surfactant was introduced into the IV dosing bag before the anti-CD79b-vc-MMAE formulation in histidine buffer to ensure that anti-CD79b-vc-MMAE as a surfactant-free ultrafiltration diafiltration (UFDF) pool was not exposed to saline in the absence of any surfactant in the IV dosing bag. The IV dosing bag was then gently rotated to allow thorough mixing while avoiding any vigorous shaking and agitation of the bag containing anti-CD79b-vc-MMAE.

[0287] Preparation of anti-CD79b-vc-MMAE in saline solution contained in glass vials: Various amounts of saline solution (0.9% sodium chloride), detergent, and immune complex were added to 15 cc Forma Vitrum glass vials and capped with 20 mm Daikyo stoppers. Samples were then incubated under the specific study conditions.

[0288] Sample Collection: Throughout the course of the study, samples were removed using 1 cc or 5 cc BD Falcon syringes coupled with 18G needles. Collected samples were stored in 10 cc PETG (Nalgene) containers.

[0289] Protein concentration determination by UV spectrophotometry scan: Protein concentrations after dilution were determined for selected samples using an Agilent spectrophotometer. Samples retrieved from the IV dosing bag were volumetrically diluted (if necessary) so that the UV signal was 0.1-1.0 AU. Absorbance was recorded at 279 nm and 320 nm. UV concentration determinations were calculated by using an extinction coefficient of 1.40 (mg / mL)-1 cm-1. Corrected A279 was obtained by subtracting A320 nm from A279 nm. This correction accounts for the turbidity of the solution and allows for accurate measurement of protein concentration.

[0290] Turbidity: The turbidity of the samples was measured in an Agilent spectrophotometer by recording the average UV absorbance from 340-360 nm using a 1 cm pathlength cuvette. The spectrophotometer was blanked with purified water.

[0291] Visible particles: The presence of any visible particles was assessed visually against a light box with a black and white background for samples in 10 cc PETG containers. Soluble aggregate content, as a measure 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 at 280 nm and a Tosoh Bioscience LLC (Montgomeryville, Pennsylvania) TSK-Gel G3000SWXL size-exclusion column (300×7.8 mm, 5 μm) at ambient temperature. Samples were eluted over 30 min with an isocratic flow rate of 0.5 mL / min using 0.2 M K3PO4, 0.25 M KCl (pH 6.2).

[0292] High Accuracy Liquid Particle Counter (HIAC): Sizing and counting of particulates while in solution was performed using the HIAC. Quantification of visible and / or subvisible particles (SVPs) used light obscuration using the HIAC (Model 9703+) instrument. 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.

[0293] Stability studies in IV administration bags during static storage: For surfactant studies in IV dosing bags, initial studies were performed under both static and stirred conditions. The dose range of anti-CD79b-vc-MMAE tested in these studies was designed and performed in the range of 1.8 mg / kg to 2.4 mg / kg. It was assumed that the weight of most patients was between 40 kg and 120 kg. Based on these assumptions, the total dose per IV dosing bag was varied from 72 mg (low dose / low body weight) to 288 mg (high dose / high body weight).

[0294] Figures 1-2 summarize the results of IV dosing bag performance with a fixed PS20 in-bag concentration of 0.005% and a final anti-CD79b-vc-MMAE concentration of 0.65 mg / mL. The concentration of anti-CD79b-vc-MMAE in the IV dosing bag was selected assuming a low dose of 1.8 mg / kg in a 100 mL IV dosing bag for a minimum patient weight of 40 kg. Since the 100 mL IV dosing bag has a pre-fill of 10 mL, the final concentration in the IV dosing bag was (1.8 mg / kg x 40 kg) / 110 mL = 0.65 mg / mL.

[0295] There were no significant differences between IV dosing pouch types regarding the physical stability of anti-CD79b-vc-MMAE under static storage conditions at 30° C. for 22 hours. All pouches showed a 0.3% + 0.1% increase (Δ) in high molecular weight species (HMWS) as measured by SEC. This demonstrated that anti-CD79b-vc-MMAE is unstable at 30° C. upon dilution in 0.9% NaCl.

[0296] Screening studies with surfactants and static storage: A well-controlled and efficient screening of surfactant types and concentrations required the use of a fixed protein concentration in the bag. A concentration of 2.6 mg / mL of anti-CD79b-vc-MMAE in the bag was selected based on the highest possible dose of 2.4 mg / kg administered in a 100 mL bag (the recommended bag size for clinics, with a 10% prefill volume) for a high-weight patient of 120 kg, as seen in the following calculation: (2.4 mg / kg x 120 kg) / 110 mL = 2.6 mg / mL. The highest protein concentration in the bag was assumed as the worst case, since a higher bulk protein concentration would promote more rapid adsorption of the antibody to the air-water interface, resulting in higher levels of interface-mediated aggregation.

[0297] Effect of temperature on stability under static storage: Providing proper storage conditions for compounded IV administration bags is important for product quality of antibody therapeutics and was evaluated. This information is also required for preparation of pharmaceutical manuals and product inserts to ensure proper use of any antibody therapeutic. Various levels of PS20 in the bags were tested at temperatures of 30°C, 25°C, and 2-8°C. At 2-8°C, the tested levels of PS20 in the bags (0.003%, 0.005%, and 0.01%) did not show significant changes in HMWS after 22 hours of static storage (Figure 3). All changes (HMWS%) were less than 0.02%. At 25°C and with PS20 levels of 0.005%, 0.01%, 0.015%, 0.025%, and 0.030% in the bags, 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 even greater than that observed at 25° C. Increases of 0.3+0.1% were observed at PS20 levels of 0.004%, 0.015%, 0.02%, 0.025%, 0.05%, and 0.1% in the bags after 22 hours of static storage (FIG. 3).

[0298] Across all temperatures, the increase in HMWS was independent of the concentration of PS20 in the pouch, indicating that aggregation under static conditions is not mediated by interfacial stress. The increase in HMWS showed a strong correlation to storage temperature. These results correlate well with a subsequent 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 shows that the formation of HMWS in antibody drug conjugates (ADCs) at higher temperatures is due to an increased sensitivity of the tertiary structure to thermal stress, possibly due to the presence of conjugated drug at the interchain cysteines near the CH2 domain.

[0299] Testing of surfactant hybrids: Poloxamer 188 (P188) is known to reduce the formation of HMWS in IV dosing bags, however, there can be a significant increase in subvisible particles (SVPs) upon agitation. In contrast, PS20 does not increase SVPs. To further explore these differing results between the two surfactants, an equal weight ratio of 1:1 PS20-P188 hybrid was used to determine if PS20 could prevent SVP formation and if P188 could limit the increase in HMWS. This study was performed in a 25 mL IV dosing bag using an anti-CD79b-vc-MMAE concentration of 2.6 mg / mL in the bag. Based on mass, equal amounts of PS20 and P188 were added to obtain a total surfactant concentration in the IV dosing bag of either 0.05% or 0.1%. In Table 1 below, under static conditions at 30°C for 22 hours, an increase in HMWS of 0.3 to 0.4% was observed. Table 1: Hybrid PS20-P188 surfactant study under thermally stressed 30°C static conditions TIFF2025032097000021.tif138170

[0300] There was a slight increase in particles sized 5 μm, 10 μm, and greater than 25 μm. When 0.1% total surfactant was used in the IV administration bag, there was an increase in 2 μm particles. Based on these results, the surfactant hybrid did not demonstrate a substantial difference over PS20 alone.

[0301] In this study, four different surfactants were evaluated, including PS20, PS80, P188, and octyl glucoside (OG). After 22 hours of incubation at 30°C with 0.02% surfactant and 2.6 mg / mL drug product in saline-filled vials from PVC bags, there was an increase in HMWS of 0.3-0.4%, demonstrating that aggregation was also independent of the type of surfactant used (Figure 4).

[0302] Stability of Anti-CD79b-vc-MMAE under static storage in vials: Considering the physical instability of anti-CD79b-vc-MMAE in normal saline (0.9% NaCl) under static storage in an IV dosing bag, the stability of the immunoconjugate in 5% dextrose (D5W) and half saline (0.45% NaCl w / v) was tested in vials to evaluate the dependency of the immunoconjugate instability on ionic strength. A solution containing 0.02% PS20 was prepared at a fixed anti-CD79b-vc-MMAE concentration of 2.6 mg / mL. Anti-CD79b-vc-MMAE showed significant improvement in physical stability both in half saline and in D5W (Figure 4). In the D5W solution, there was no change in HMWS, while the half saline solution showed an increase in HMWS of less than 0.1% after 24 hours of static storage at 30°C. Although preparation in D5W limited aggregation of anti-CD79b-vc-MMAE, glycation of antibodies in D5W solutions 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, IV delivery of anti-CD79b-vc-MMAE in 5% dextrose pouches was not further considered due to the potential for glycation. Although half saline showed significant improvement in physical stability compared to normal saline, the availability of pre-made half saline pouches across clinical and commercial settings was low, and the inconvenience and potential microbial contamination risk associated with preparation of half saline pouches in pharmacies also led to the use of half saline for IV infusion of anti-CD79b-vc-MMAE being disfavored.

[0303] Conclusions from static stability studies in IV dosing bags and glass vials: The static stability studies described above demonstrate that aggregation of anti-CD79b-vc-MMAE is promoted by a high ionic strength environment, showing that the aggregation rate in the formulation buffer is significantly slower than that in saline.

[0304] B. IV Dose Bag Agitation Studies: A series of IV dosing bag agitation studies were performed to support the transportation of compounded IV dosing bags containing anti-CD79b-vc-MMAE for dose administration in hospital or clinical settings. Although transportation of prepared IV dosing bags containing therapeutic proteins is generally not recommended due to aggregation risk, there is often a practical need to transport IV dosing bags in clinical settings, especially in remote areas with limited access to pharmacies. Therefore, minimal transportation should be supported, and the impact of IV dosing bag agitation on the drug must be evaluated during development. Agitation stress within the IV dosing bag during transportation can induce physical degradation to the therapeutic antibody. Physical degradation of the antibody (i.e., aggregation and particle formation) may be mediated by adsorption to the air-liquid interface within the IV dosing bag, which undergoes continuous regeneration during agitation. Throughout the course of the following study, a MaxQ4000 elliptical laboratory shaker (0.75 inch orbit length) was utilized for the IV dosing bag agitation studies. The IV dosing bag was placed flat on the flat surface of the shaker.

[0305] Effect of temperature on agitation stability in IV dosing bags: Agitation studies were performed at 2-8°C, 25°C, and 30°C with different levels of PS20 in the bag (Figure 5A-C). The rate of increase in HMWS was faster at 30°C than at 25°C, which was faster than those tested at 2-8°C. The increase in HMWS correlated to the temperature in the IV dosing bag during agitation. For bags containing PS20 concentration levels of 0.01%, 0.005%, and 0.003%, there was no increase in HMWS upon agitation for 2 hours at 2-8°C (Figure 5A). At 30°C, the increase in HMWS was dependent on the concentration of PS20 in the bag (Figure 5C). Note that the increase in HMWS upon agitation was nonlinear across all PS20 levels at both 2-8°C (Figure 5A) and 25°C (Figure 5B). This nonlinear behavior could be due to sampling at different time points.

[0306] Effect of surfactant mixtures on agitation stability in IV dosing bags: An equal 1:1 weight ratio of PS20-P188 hybrid was used to determine whether PS20 could prevent SVP formation and P188 could limit the increase in HMWS upon agitation. This study was performed in a 25 mL IV dosing bag using an anti-CD79b-vc-MMAE concentration of 2.6 mg / mL in the bag. Equal amounts of PS20 and P188, based on mass, were added to obtain a total surfactant concentration in the IV dosing bag of either 0.05% or 0.1%.

[0307] When 0.05% total surfactant was used in the IV dosing bag, an increase in HMWS of 2.9% was observed after 2 hours of stirring at 30° C. (Table 2). There was a trend for an increase in particles of sizes 5 μm, 10 μm, and greater than 25 μm up to 1 hour, with particle counts exceeding the limit of detection (LOD) at 2 hours. When 0.1% total surfactant was used in the IV dosing bag, there was a trend for an increase in particles across all sizes, but was significantly lower than the bag with 0.05% surfactant (Table 2). The increase in HMWS was also lower by about 2.8% after 2 hours of stirring compared to the total surfactant concentration in the IV dosing bag at 0.05%. Based on these results, the surfactant hybrid showed no difference versus PS20 alone. Table 2: Hybrid PS20-P188 surfactant study under heat stressed 30 °C stirring conditions TIFF2025032097000022.tif114170

[0308] Short-term IV bag agitation at 30°C: The purpose of this study was to investigate the transportation of IV dosing bags with diluted anti-CD79b-vc-MMAE at ambient conditions for a short period of time and to determine the effective concentration of PS20 in the anti-CD79b-vc-MMAAE formulation. The study subjects the bags to a short period of agitation for 30 minutes at 30° C. This shorter agitation at 30° C. may encompass potential agitation that diluted anti-CD79b-vc-MMAE may experience during transportation at ambient conditions in a hospital or other facility. The study was performed with 0.001%, 0.002%, 0.003%, and 0.005% PS20 diluted in an IV dosing bag and agitated at 30° C. and 100 rpm. The results are shown in FIG. 6. Based on these results, with 0.005% PS20 in the bag, there is an increase of less than 0.1%. Under these conditions, for anti-CD79b-vc-MMAE, there is a direct correlation between the increase in HMWS and the concentration of PS20 in the IV dosing bag. Based on this study, regarding the physical stability of diluted anti-CD79b-vc-MMAE in an IV dosing bag, the minimum amount of PS20 in the IV dosing bag to maintain aggregation levels below the limit of quantification was 0.003%-0.005%.

[0309] Agitation during cooling of IV administration bag at 2-8°C (without pre-cooling bag): The purpose of this study was to support the transportation of compounded IV dosing bags in a more realistic scenario. Typically, IV dosing bags are compounded by a pharmacist and transported in a vehicle that may or may not be pre-cooled. Prior to the start of this experiment, the bags were equilibrated to 30 °C to simulate the higher ambient temperatures that may exist in different regions around the world. This ensures the stability of the diluted DP even if the bag is not pre-cooled or is transported in a non-pre-cooled container. This study was performed with 2.6 mg / ml anti-CD79b-vc-MMAE with four different 100 mL PS20 levels (0.001%, 0.002%, 0.003%, and 0.005%) in the IV dosing bag. There is less than 0.1% increase in HMWS (less than 0.002% in the IV dosing bag). Based on the results of this agitation model study, the minimum amount of PS20 in the IV dosing bag to maintain aggregation levels below the limit of quantification was 0.001%-0.002%. The results are summarized in FIG.

[0310] Conclusion: Antibody-drug conjugates (ADCs) are particularly sensitive to saline and high ionic strength buffers compared to conventional unconjugated antibodies (Adem, Y., et al. Bioconjugate Chemistry, 25 (2014) 656-664). The anti-CD79b-vc-MMAE tested demonstrated an increase in HMWS upon dilution in 0.3% saline under static storage at 30°C for 22 hours.

[0311] Based on two agitation models, anti-CD79b-vc-MMAE is stable at at least 0.002% PS20 in the IV dosing bag during agitation induced by short-term transport at 2-8 °C for 1 h. To meet the stability requirements against agitation stress during handling and transport as well as short-term storage, the final proposed surfactant concentration in the anti-CD79b-vc-MMAE formulation is 0.12%, which when combined with the use of a 100 mL IV dosing bag results in 0.004% in the bag for the lowest expected patient weight (40 kg). This level of surfactant was shown to protect anti-CD79b-vc-MMAE for a dose range of 1.8 mg / kg to 2.4 mg / kg. It should be noted that this amount of surfactant, particularly PS20, is unexpectedly high and counterintuitive in light of the scientific literature (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). This amount of PS20 used in the anti-CD79b-vc-MMAE drug product is also unexpectedly higher compared to another immunoconjugate, anti-CD22-vc-MMAE, for which a commercial formulation of 0.05% PS20 has been established as suitable for dilution into IV dosing bags (data not shown).

[0312] Example 2: Mitigating the risk of anti-CD79b immunoconjugate oxidation and linker hydrolysis Establishing a suitable surfactant concentration for IV administration bag delivery as discussed in Example 1 potentially increased the problem of oxidation of anti-CD79b-vc-MMAE. It is known that higher levels of polysorbate are generally associated with higher levels of peroxides due to autooxidative degradation of polysorbate. 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).

[0313] Furthermore, it is known that tryptophan oxidation can 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. Of note, in the anti-CD79b antibody used in this disclosure, tryptophan is present in the variable heavy chain HVR1:GYTFSSYWIE (SEQ ID NO: 4). The tendency to oxidize under AAPH stress conditions (e.g., at 40°C for up to 2 weeks or at 25°C for 6 months or at 2-8°C for 6 months) was determined by mass spectrometry analysis of tryptic peptides after formulation of anti-CD79b-vc-MMAE with AAPH. Stressed anti-CD79b-vc-MMAE was digested with trypsin and the digested peptides were subjected to LC-MS / MS to determine the percentage of oxidation (data not shown). By peptide mapping, 68% oxidation of HVR tryptophan was observed, which resulted in approximately 58% loss of potency for anti-CD79b-vc-MMAE (data not shown). Therefore, it is necessary to mitigate the oxidation risk.

[0314] Additional factors to consider included how the immunoconjugate's linker would handle succinimide hydrolysis. The present anti-CD79b-vc-MMAE immunoconjugate employed a maleimide-containing drug-linker that was conjugated to an antibody cysteine ​​residue to form a thio-succinimide bond. It is known that such thio-succinimide bonds can undergo two competing reactions in patient plasma: (a) removal of the maleimide, resulting in unwanted loss of drug from the immunoconjugate, and (b) hydrolysis of the thio-succinimide ring, resulting in a succinic acid derivative that cannot undergo removal. 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) as it has potential impacts on pharmacokinetics (pK) and safety. Initial liquid formulations used in early phase clinical trials in patients, e.g., liquid formulations in histidine acetate buffer (pH 5.5), have previously been demonstrated to demonstrate an approximately 9% increase in acidity over a 2-year period at recommended storage conditions (data not shown).

[0315] As a result of these three risks, namely increased oxidation risk due to increased PS20 concentration, increased oxidation risk due to tryptophan oxidation resulting in loss of potency, and linker hydrolysis risk resulting in loss of MMAE drug from the immune complex and resulting in free drug in the patient's plasma, a lyophilized formulation was proposed to mitigate these risks and provide a longer and more stable shelf life.

[0316] A. Investigation of alternative buffer types: To begin formulation development of the lyophilized product, alternative buffer species were investigated. The buffer species used for the early clinical phase formulation was histidine acetate buffer. L-histidine is known to have oxidation potential through a variety of mechanisms, of which the most common oxidation mechanism observed is photooxidation. See Mason, B., et al Oxidation of Free L-histidine by tert-Butylhydroperoxide., Pharm Res. (2010) 27(3): 447-456. In addition, acetic acid can evaporate during the lyophilization process, thereby resulting in a change in pH that may destabilize the immunoconjugate product. Furthermore, since acetic acid is flammable, the inclusion of acetic acid may be considered a potential hazard to the lyophilization process. Therefore, alternative buffer species were evaluated. Three formulations shown in Table 3 were tested. Table 3: Buffer type screening and pH range evaluation TIFF2025032097000023.tif37170

[0317] The stability of frozen-thawed anti-CD79b-vc-MMAE pharmaceutical compositions of Table 3 in disposable PETG containers was evaluated in a freeze / thaw stress experiment. The protein formulation was sterile filtered and filled into PETG containers. The containers were placed at -20°C for T=0 and 1 week. The containers were frozen and thawed to room temperature a total of 5 times. Only T=0 and the 3rd freeze-thaw cycle were analyzed. The results are summarized in Table 4. Table 4: Stability of anti-CD79b-vc-MMAE after freezing and thawing TIFF2025032097000024.tif100170

[0318] These results demonstrate that no problems were observed with any of the three formulations tested. Therefore, the use of histidine or succinic acid would be acceptable alternative buffer species to histidine acetate.

[0319] Next, a stability screen was tested, testing the effect of pH on charge variant formation at 30°C. The same three liquid formulations as in Table 3 were used and stressed at 30°C for three different time points: 2 weeks, 4 weeks, and 8 weeks, and charge variant distribution was evaluated by an iCE280 analyzer (ProteinSimple) with a PrinCE microsyringe and a 100 μm x 5 cm fluorocarbon-coated capillary cartridge (ProteinSimple). To remove the heavy chain C-terminal lysine residues, carboxypeptidase B (CpB) was added to each sample after the dilution step at an enzyme-to-substrate ratio of 1:1000 (wt / wt). In addition, 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 samples were mixed with an ampholyte solution consisting of a mixture of 700 μL of 1% methylcellulose, 1218 μL of purified water, 8 μL of Pharmalyte 8-10.5, 55 μL of Pharmalyte 5-8, 15 μL of pI marker 5.12, and 4 μL of pI marker 7.05. The samples were focused by introducing a potential of 1500 V for 1 min, followed by a potential of 3000 V for 5 min, using an anolyte of 80 mM phosphoric acid and a catholyte of 100 mM sodium hydroxide, both in 0.1% methylcellulose. Images of the focused charge variants were obtained by passing 280 nm ultraviolet (UV) light through the capillary and into the lens of a charge-coupled device digital camera. The results are shown in Figures 9 and 10, which demonstrate that at pH 5.0-5.5, there is less formation of acidic species and slightly more rapid formation of basic species. Thus, the buffer components do not appear to have a measurable effect on charge stability.

[0320] B. Investigating the effect of pH on stability: The same three liquid formulations in Table 3 were stressed at 30°C at three different time points, 2 weeks, 4 weeks, and 8 weeks, and a stability screen was tested to test the effect of pH on size variant formation at 30°C. Size variant distribution was determined by size exclusion chromatography (SEC) on an Agilent Technologies 1200 series HPLC (Santa Clara, California) using 0.25 mM potassium chloride (pH 6.2, mobile phase). Liquid formulation samples were then loaded onto a Tosoh Bioscience TSKgel G3000SWXL column (South San Francisco, California). Samples were eluted over 30 minutes using a flow rate of 0.5 ml / min and absorbance at 280 nm was monitored. Results are reported as relative peak area of ​​total area under the curve and are shown in Figure 11. The stability screen demonstrated that less high molecular weight species (HMWS) were observed at pH 5.0-5.5 compared to pH 6.0. Similar amounts of low molecular weight species (LMWS) were observed after 4 weeks at 30° C. Slightly higher LMWS was observed at 8 weeks at 30° C. A decrease in pH to 5.3 appeared to reduce both charge and size variant formation, so pH 5.3 was chosen.

[0321] Figure 12 shows chromatograms testing two buffer types, histidine / histidine-HCl (pH 5.5), in comparison with sodium succinate at three different pHs (5.0, 5.5, and 6.0). The histidine buffer at pH 5.5 showed slightly better physical stability in liquid at 30°C than sodium succinate at all pHs tested (pH 5.0, 5.5, and 6.0). However, sodium succinate buffer was chosen because it showed acceptable stability in liquid, considering the potential risk of oxidation of histidine.

[0322] C. Testing of Lyophilized Formulations: To mitigate the above mentioned risks, a lyophilized formulation was tested. The general scheme of the lyophilization cycle is shown in Table 5. Table 5: Lyophilization cycle for anti-CD79b-vc-MMAE TIFF2025032097000025.tif72170

[0323] The cycle length was approximately 78 hours or about 3 days. The moisture content was set at 0.5% to 0.7%.

[0324] A lyophilization screening study was performed testing various anti-CD79b-vc-MMAE concentrations, two different buffer types, different pH, and two different surfactant levels, as shown in Table 6. Table 6: Lyophilization Screening Study - Test Formulations TIFF2025032097000026.tif77170

[0325] These test lyophilized formulations were stored at 30° C. for up to 2 months as part of the screening study.

[0326] SEC analysis was used after reconstitution of each formulation tested to 10 mg / ml or 20 mg / ml as described above to quantify any monomers developed for each formulation tested (1)-(5) as an indicator of stability. Figure 13 summarizes this study and demonstrates that there was no change in the percent monomer in any of the formulations tested over a 2-month period of heat stress at 30°C. Each lyophilized formulation (1)-(5) was also tested by icIEF as described above to determine the percent main peak after 2 months of heat stress at 30°C. Figure 14 summarizes this study and demonstrates that there was no change in the percent main peak in any of the formulations tested over a 2-month period of heat stress at 30°C. As a result of these two studies, it was determined that sodium succinate buffers over a pH range of 5.0-6.0 provided acceptable stability for the lyophilized formulations tested.

[0327] D. Appearance of the freeze-dried cake: One important aspect of a lyophilized formulation is the actual appearance of the lyophilized cake, since appearance is a product quality attribute. For lyophilized formulations of anti-CD79b-vc-MMAE, both immunoconjugate concentration and stabilizer / tonicity agent concentration were tested to examine ways to improve the appearance of the lyophilized cake. Figures 15A-C show the appearance of three lyophilized formulations with different protein-to-sucrose ratios tested. For all three sucrose levels tested, nicks were observed at the bottom of the lyophilized cake, which is not the ideal appearance of the lyophilized cake. Nevertheless, under similar lyophilization conditions, the formulation with less sucrose showed smaller nicks, and lower sucrose concentrations result in less moisture. Another lyophilized formulation was tested in which 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 cakes. The combination of 20 mg / ml immunoconjugate concentration and 120 mM sucrose demonstrated the most robust and homogenous lyophilized cake appearance. No changes in cake structure, color, or appearance were observed when heat stress stability studies were performed over a 4-week period at 40° C., 25° C., and 5° C., and moisture content remained below 5%.

[0328] E. Stability of Lyophilized Formulations: A lyophilized formulation of the drug product: 20 mg / ml anti-CD79b-vc-MMAE, 120 mM sucrose, 0.12% PS20 in 10 mM sodium succinate, pH 5.3, was tested for stability as measured by soluble aggregate or charge variant formation. Soluble aggregates were measured by SEC as described above. Figure 17 shows that there was less than a 0.1% increase in soluble aggregates over 8 weeks at 25°C and 2-8°C for the lyophilized drug product. At 40°C, an increase of approximately 0.1% of dimeric aggregate species per week was observed, and this aggregation rate may be dependent on moisture levels. Charge variants were measured by icIEF as described above. Figure 18 shows that for the lyophilized drug product, there was approximately a 5% loss of the main peak and a 3.7% increase in acidic species after 4 weeks at 40°C. There was no significant change measured at 25°C and 2-8°C. As a result, no changes were observed in the drug product freeze-dried under stress conditions at 25°C and 2-8°C.

[0329] Table 7 summarizes the final anti-CD79b-vc-MMAE formulation that was arrived at as a result of the extensive work described in Examples 1 and 2. Table 7: Summary of final anti-CD79b-vc-MMAE formulations. TIFF2025032097000027.tif101170

[0330] As shown in Table 7 above, the final commercial drug product formulation contains 20 mg / ml of drug product, which increases the protein to drug ratio and results in improved cake appearance upon lyophilization of the solution. The 0.12% PS20 (i.e., 1.2 mg / ml) protects the protein in the formulation from interfacial stresses encountered during administration using IV administration bags and IV infusion sets. The pH of 5.3 minimizes the formation of acidic variants contributed by succinimide hydrolysis. The formulation in Table 7 was designed to deliver 140 mg of drug product per vial after reconstitution in 7.2 mL of SWFI to support a clinical dose of 1.8 mg / kg.

[0331] Example 3: Evaluation of the robustness of anti-CD79b-vc-MMAE drug substance and drug product formulation The drug product in the lyophilized formulation (see Table 7) was found to be stable for at least 44 months at 2°C to 8°C over a residual moisture content range of 0.3% to 3.2% by weight, and for at least 7 months at 25°C over a residual moisture content range of 0.3% to 3.2% by weight.

[0332] Next, formulation parameters of the lyophilized drug product were varied and evaluated for practically meaningful effects on the stability of the lyophilized drug product. The five formulation parameters tested (and the range over which the parameters were varied) were: (a) protein concentration (17-23 mg / ml), (b) succinic acid 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). No practically meaningful effects on the stability of the lyophilized drug product were observed for up to 9 months after storage at 2°C-8°C. Based on the model predicted degradation rates, no practically meaningful effects on the stability of the lyophilized drug product are expected for up to at least 24 months after storage at 2°C-8°C.

[0333] The reconstituted drug product solution (i.e., 20 mg / mL polatuzumab vedotin in 10 mM succinic acid, 120 mM sucrose, 1.2 mg / mL polysorbate 20, pH 5.3) was found to be physicochemically stable after 72 hours of storage at 2 °C-8 °C or after 24 hours of storage at 30 °C with exposure to ambient light. No significant changes (e.g., statistically significant changes) were detected in the affinity of polatuzumab vedotin for its target (i.e., CD79b) or in the biological activity of polatuzumab vedotin.

[0334] The present disclosure has been described in some detail by way of illustrations and examples for clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

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

2. 2. The pharmaceutical composition of claim 1, wherein the anti-CD79b immunoconjugate is at a concentration of 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. 3. The pharmaceutical composition of claim 2, wherein the anti-CD79b immunoconjugate is at a concentration of about 10 mg / ml to about 20 mg / ml.

4. 4. The pharmaceutical composition of claim 2 or 3, wherein the anti-CD79b immunoconjugate is at a concentration of 20 mg / ml.

5. The pharmaceutical composition of any one of claims 1 to 3, wherein the composition comprises the anti-CD79b immunoconjugate at a concentration of 10 mg / ml and the surfactant at a concentration of 0.06% weight / volume.

6. 5. The pharmaceutical composition of claim 1, wherein the composition comprises the anti-CD79b immunoconjugate at a concentration of 20 mg / ml and the surfactant at a concentration of 0.12% weight / volume.

7. 5. The pharmaceutical composition of claim 1, wherein said composition comprises said anti-CD79b immunoconjugate at a concentration of 20 mg / ml and said surfactant at a concentration of at least 0.12% weight / volume.

8. 8. The pharmaceutical composition of claim 1, wherein 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 of any one of claims 1 to 8, wherein the composition further comprises a buffering agent.

10. 10. The pharmaceutical composition of claim 9, wherein the buffer is a histidine buffer or a succinate buffer.

11. The pharmaceutical composition of claim 10, wherein the buffer is a histidine buffer.

12. 11. The pharmaceutical composition of claim 10, wherein the buffer is a succinate buffer.

13. The composition of claim 12, wherein the succinate buffer is a sodium succinate buffer.

14. 14. The pharmaceutical composition of claim 13, wherein the sodium succinate buffer is at a concentration of about 10 mM to about 200 mM.

15. 15. The pharmaceutical composition of claim 14, wherein the sodium succinate buffer is at a concentration of 10 mM.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the composition has a pH of about 5.0 to about 6.

0.

17. 17. The pharmaceutical composition of claim 16, wherein the composition has a pH of 5.

3.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the composition further comprises a sugar.

19. 20. The pharmaceutical composition of claim 18, wherein the sugar is at a concentration of about 100 mM to about 260 mM.

20. The pharmaceutical composition according to claims 18 to 19, wherein the sugar is selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, dextran 40, and trehalose.

21. 21. The pharmaceutical composition of claim 20, wherein the sugar is sucrose.

22. 23. The pharmaceutical composition of claim 22, wherein the sucrose is at a concentration of 120 mM.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the composition is reconstituted from a lyophilized cake.

24. A pharmaceutical composition produced by lyophilization of a liquid composition containing 20 mg / ml of an anti-CD79b immune complex, 0.12% w / v of polysorbate 20, and 120 mM of sucrose in 10 mM sodium succinate buffer, wherein the liquid composition has a pH of 5.3, and the anti-CD79b immune complex has the formula, including, wherein, Ab is an anti-CD79b antibody, the anti-CD79b antibody includes a heavy chain and a light chain, the light chain includes (a) the HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) the HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) the HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), the heavy chain includes (a) the HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) the HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) the HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, p is a value from about 1 to about 8, the pharmaceutical composition.

25. The pharmaceutical composition according to any one of claims 1 to 24, wherein the anti-CD79b antibody includes a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO:

8.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the heavy chain includes the amino acid sequence of SEQ ID NO: 9 and the light chain includes the amino acid sequence of SEQ ID NO:

10.

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein p is a value from about 2 to about 5.

28. A pharmaceutical composition produced by lyophilization of a liquid composition containing 20 mg / ml of an anti-CD79b immune complex, 0.12% w / v of polysorbate 20, and 120 mM of sucrose in 10 mM sodium succinate buffer, wherein the liquid composition has a pH of 5.3, and the anti-CD79b immune complex has the formula, including, wherein, Ab is an anti-CD79b antibody, the anti-CD79b antibody includes a heavy chain and a light chain, the heavy chain includes the amino acid sequence of SEQ ID NO: 9, the light chain includes the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, and p is a value from about 2 to about 5, the pharmaceutical composition.

29. The pharmaceutical composition of any one of claims 1 to 28, wherein p is about 3.

5.

30. 30. The pharmaceutical composition of any one of claims 24 to 29, wherein the composition has a stability of about 48 months at 5°C ± 3°C when protected from light.

31. 31. The pharmaceutical composition of claim 30, wherein the stability of the composition is measured by size-exclusion high performance liquid chromatography (SE-HPLC).

32. 32. The pharmaceutical composition of claim 31, wherein the composition has a main peak area % of at least 95.0 as measured by SE-HPLC.

33. The pharmaceutical composition of any one of claims 28 to 32, wherein the stability of the composition is measured by imaging capillary isoelectric focusing (icIEF).

34. 34. The pharmaceutical composition of claim 33, wherein the composition has a main peak (area%) of at least 58.0, an acidic area (area%) of at most 32.0, and a basic area (area%) of at most 12.0 as measured by icIEF.

35. The pharmaceutical composition according to any one of claims 25 to 34, wherein the pharmaceutical composition is a lyophilized cake.

36. The pharmaceutical composition of any one of claims 1 to 35, wherein the composition is reconstituted with sterile water for injection (SWFI).

37. 37. The pharmaceutical composition of claim 36, wherein the composition is reconstituted in about 7.2 ml of SWFI.

38. 38. The pharmaceutical composition of claim 37, wherein the reconstituted composition is stable for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours upon storage at about 30°C.

39. 39. The pharmaceutical composition of claim 37 or claim 38, wherein the reconstituted composition is stable for at least 24, 48, or 72 hours upon storage at about 2°C to about 8°C.

40. 40. The pharmaceutical composition of any one of claims 36 to 39, wherein the reconstituted composition is further diluted in an isotonic buffer in an intravenous (IV) administration bag.

41. 41. The pharmaceutical composition of claim 40, wherein the final volume of the diluted composition in the IV administration bag is about 100 ml.

42. 42. The pharmaceutical composition of claim 40 or 41, wherein the concentration of the immunoconjugate in the IV administration bag is from about 0.72 mg / ml to about 2.7 mg / ml.

43. 1. A pharmaceutical composition produced by lyophilization of a liquid composition comprising 20 mg / ml anti-CD79b immunoconjugate, 0.12% by weight / volume polysorbate 20, and 120 mM sucrose in 10 mM sodium succinate buffer, said liquid composition having a pH of 5.3, and said anti-CD79b immunoconjugate having the formula: wherein Ab is an anti-CD79b antibody, said anti-CD79b antibody comprising a heavy chain and a light chain, said heavy chain comprising the amino acid sequence of SEQ ID NO:9, said light chain comprising the amino acid sequence of SEQ ID NO:10, Val is valine, Cit is citrulline, and p has a value of about 2 to about 5; The composition is reconstituted with about 7.2 ml of sterile water for injection (SWFI) and further diluted in an isotonic buffer in an intravenous (IV) administration bag; the final volume of the diluted composition in the IV administration bag is about 100 ml; The pharmaceutical composition, wherein the final concentration of the immunoconjugate in the IV administration bag is about 0.72 mg / ml or about 2.7 mg / ml.

44. 44. The pharmaceutical composition of claim 43, wherein p is about 3.

5.

45. 45. The pharmaceutical composition of any one of claims 38 to 44, wherein the isotonic buffer is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, or a 5% dextrose solution.

46. 1. A liquid composition comprising: a) about 0.72-2.7 mg / ml of polatuzumab vedotin; b) about 0.36-01.35 mM of sodium succinate; c) about 0.51-16.24 mM of sucrose; and d) about 0.0432-0.162 mg / ml of polysorbate 20, wherein the pH of the liquid composition is about 5 to about 5.

7.

47. 1. 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; and d) about 0.0432 mg / ml polysorbate 20, wherein the pH of the liquid composition is 5.1 to about 5.

4.

48. 1. A liquid composition comprising: a) about 2.7 mg / ml polatuzumab vedotin; b) about 0.1.35 mM sodium succinate; c) about 16.24 mM sucrose; and d) about 0.162 mg / ml polysorbate 20, wherein the pH of the liquid composition is about 5.1 to about 5.

4.

49. The liquid composition according to any one of claims 46 to 48, wherein the volume of the liquid composition is from about 50 ml to about 100 ml.

50. 50. The liquid composition of claim 49, wherein the volume of the liquid composition is 50 ml.

51. 50. The liquid composition of claim 49, wherein the volume of the liquid composition is 100 ml.

52. 1. A pharmaceutical composition comprising an anti-CD79b immune conjugate, a surfactant, a succinate buffer, and a sugar, wherein said pharmaceutical composition, when reconstituted in water, forms a liquid pharmaceutical composition comprising said anti-CD79b immune conjugate at a concentration of about 10 mg / ml to about 20 mg / ml, said surfactant at a concentration of at least 0.06% weight / volume, said succinate buffer at a concentration of about 10 mM to about 200 mM, and said sugar at a concentration of about 100 mM to about 260 mM, said liquid pharmaceutical composition having a pH of 5.3, and wherein said anti-CD79b immune conjugate has a structure represented by the formula: wherein Ab is an anti-CD79b antibody, said anti-CD79b antibody comprising a heavy chain and a light chain, said light chain comprising (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), said heavy chain comprising (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, The pharmaceutical composition wherein p is a value of about 1 to about 8.

53. 53. The pharmaceutical composition of claim 52, wherein the anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:

8.

54. 54. The pharmaceutical composition of claim 52 or 53, wherein the heavy chain of the anti-CD79b comprises the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO:

10.

55. 55. The pharmaceutical composition of any one of claims 24-34 or 52-54, wherein the pharmaceutical composition is produced by lyophilization, and the lyophilized pharmaceutical composition is stable for at least 6, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months upon storage at about 2°C to about 8°C.

56. 56. The pharmaceutical composition of claim 55, wherein the lyophilized pharmaceutical composition is a lyophilized cake.

57. 57. The pharmaceutical composition of any one of claims 52-56, wherein the pharmaceutical composition is stable upon storage at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours after reconstitution.

58. 58. The pharmaceutical composition of any one of claims 52-57, wherein the pharmaceutical composition is stable for at least 24, 48, or 72 hours upon storage at about 2°C to about 8°C after reconstitution.

59. 59. The pharmaceutical composition of any one of claims 52-58, wherein the pharmaceutical composition, after reconstitution, is then diluted into an isotonic buffer in an IV administration bag, such that the concentration of the surfactant upon dilution into the IV administration bag is at least 0.003% weight / volume.

60. 60. The pharmaceutical composition of claim 59, wherein the surfactant concentration upon dilution into the IV administration bag is at least 0.004% weight / volume.

61. 61. The pharmaceutical composition of any one of claims 52 to 60, wherein the surfactant is polysorbate 20.

62. 62. The pharmaceutical composition of any one of claims 52 to 61, wherein the sugar is sucrose.

63. 63. The pharmaceutical composition of any one of claims 52-58 and 61-62, wherein the sucrose is at a concentration of 120 mM.

64. 64. The pharmaceutical composition of any one of claims 52 to 63, wherein the succinate buffer is a sodium succinate buffer.

65. 65. The pharmaceutical composition of claim 64, wherein after reconstitution but prior to dilution, the sodium succinate buffer is at a concentration of 10 mM.

66. 66. The pharmaceutical composition of any one of claims 52-58 and 61-65, wherein the composition has a stability at 5°C ± 3°C for about 7 days.

67. 67. The pharmaceutical composition of claim 66, wherein the stability of the composition is measured by size-exclusion high performance liquid chromatography (SE-HPLC).

68. 68. The pharmaceutical composition of claim 67, wherein the composition has a main peak area % of at least 95.0 as measured by SE-HPLC.

69. The pharmaceutical composition of any one of claims 52 to 68, wherein the stability of the composition is measured by imaging capillary isoelectric focusing (icIEF).

70. 70. The pharmaceutical composition of claim 69, wherein the composition has a main peak (area%) of at least 58.0, an acidic area (area%) of up to 32.0, and a basic area (area%) of up to 12.0 as measured by icIEF.

71. A glass vial containing the pharmaceutical composition according to any one of claims 452 to 58 and 61 to 70.

72. 1. A liquid composition comprising an anti-CD79b immune conjugate, a surfactant, a succinate buffer, and a sugar, wherein the anti-CD79b immune conjugate is at a concentration of about 10 mg / ml to about 20 mg / ml, the surfactant is at a concentration of at least 0.06% weight / volume, the succinate buffer is at a concentration of about 10 mM to about 200 mM, and the sugar is at a concentration of about 100 mM to about 260 mM, the liquid composition has a pH of 5.3, and the anti-CD79b immune conjugate has a structure represented by the formula: wherein Ab is an anti-CD79b antibody, said anti-CD79b antibody comprising a heavy chain and a light chain, said light chain comprising (a) an HVR-L1 sequence of KASQSVDYEGDSFLN (SEQ ID NO: 1), (b) an HVR-L2 sequence of AASNLES (SEQ ID NO: 2), and (c) an HVR-L3 sequence of QQSNEDPLT (SEQ ID NO: 3), said heavy chain comprising (a) an HVR-H1 sequence of GYTFSSYWIE (SEQ ID NO: 4), (b) an HVR-H2 sequence of GEILPGGGDTNYNEIFKG (SEQ ID NO: 5), and (c) an HVR-H3 sequence of TRRVPIRLDY (SEQ ID NO: 6), Val is valine, Cit is citrulline, The liquid composition wherein p is a value of from about 1 to about 8.

73. 73. The liquid composition of claim 72, wherein the anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:

8.

74. 74. The liquid composition of claim 72 or 73, wherein the heavy chain of the anti-CD79b comprises the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO:

10.

75. The liquid composition according to any one of claims 72 to 74, wherein the surfactant is polysorbate 20.

76. 76. The liquid composition according to any one of claims 72 to 75, wherein the sugar is sucrose.

77. 77. The liquid composition of claim 76, wherein the sucrose is at a concentration of about 120 mM.

78. The liquid composition according to any one of claims 72 to 77, wherein the succinic acid buffer is a sodium succinate buffer.

79. 79. The liquid composition of claim 78, wherein the sodium succinate buffer is at a concentration of 10 mM.

80. The liquid composition of any one of claims 72 to 79, wherein the composition is diluted in an isotonic buffer.

81. 81. The liquid composition of claim 80, wherein the isotonic buffer is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, or a 5% dextrose solution.

82. 82. The liquid composition of claim 80 or 81, wherein the composition to be diluted in an isotonic buffer is in an IV administration bag.

83. 80. The liquid composition of any one of claims 72 to 79, wherein the composition has a stability of about 72 hours at 5°C ± 3°C.

84. 80. The liquid composition of any one of claims 72 to 79, wherein the composition has a stability of about 24 hours at 25°C.

85. 80. The liquid composition of any one of claims 72 to 79, wherein the liquid composition is stable upon storage at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours.

86. 80. The liquid composition of any one of claims 72 to 79, wherein the liquid composition is stable upon storage at about 2°C to about 8°C for at least 24, 48, or 72 hours.

87. 1. 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 has the formula: wherein Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain of the anti-CD79b comprises the amino acid sequence of SEQ ID NO: 9, the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, The lyophilized pharmaceutical composition, wherein p is a value from about 2 to about 5.

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.

89. A lyophilized pharmaceutical composition comprising about 140 mg of anti-CD79b immune complex, 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 immune complex has the formula, comprising, wherein, Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain of the anti-CD79b comprises the amino acid sequence of SEQ ID NO: 9, the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, The lyophilized pharmaceutical composition, wherein p is a value from about 2 to about 5.

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.

91. A lyophilized pharmaceutical composition comprising about 30 mg of anti-CD79b immune complex, 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 immune complex has the formula, comprising, wherein, Ab is an anti-CD79b antibody, the anti-CD79b antibody comprises a heavy chain and a light chain, the heavy chain of the anti-CD79b comprises the amino acid sequence of SEQ ID NO: 9, the light chain of the anti-CD79b antibody comprises the amino acid sequence of SEQ ID NO: 10, Val is valine, Cit is citrulline, and p is a value from about 2 to about 5, the lyophilized pharmaceutical composition.

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.

93. 92. The freeze-dried composition of any one of claims 87, 89, or 91, wherein p is about 3.

5.

94. 1. 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.

95. 95. The lyophilized pharmaceutical composition of any one of claims 87-94, wherein the lyophilized pharmaceutical composition is stable for at least 6, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months upon storage at about 2°C to about 8°C after reconstitution.

96. 96. The lyophilized pharmaceutical composition of any one of claims 87 to 95, wherein the lyophilized pharmaceutical composition is a lyophilized cake.

97. 1. A liquid pharmaceutical composition comprising: a) 5-60 mg / ml polatuzumab vedotin; b) 10-200 mM sodium succinate; c) 100-260 mM sucrose; and d) 0.06-0.12% by weight or volume of polysorbate 20, wherein the pH of the liquid composition is 5-6.

98. 98. The liquid composition of claim 97, comprising: a) 10-55 mg / ml polatuzumab vedotin; b) 10-100 mM sodium succinate; c) 150-260 mM sucrose; d) 0.08-0.12% by weight / volume polysorbate 20; and wherein the pH of the liquid composition is 5.1-5.

6.

99. 98. The liquid composition of claim 97, comprising: a) 15-40 mg / ml polatuzumab vedotin; b) 10-50 mM sodium succinate; c) 200-260 mM sucrose; d) 0.1-0.12% by weight / volume polysorbate 20; and wherein the pH of the liquid composition is 5.2-5.

4.

100. 1. A liquid pharmaceutical composition comprising: a) 20 mg / ml polatuzumab vedotin; b) 10 mM sodium succinate; c) 120 mM sucrose; d) 0.12% by weight or volume polysorbate 20; and wherein the pH of the liquid composition is about 5.

3.

101. 101. The liquid composition of any one of claims 97 to 100, wherein the liquid composition is stable upon storage at about 30°C for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours.

101. 102. The liquid composition of any one of claims 97 to 101, wherein the liquid composition is stable upon storage at about 2°C to about 8°C for at least 24, 48, or 72 hours.

102. 13. A method of treating a proliferative disorder in a patient in need thereof, comprising administering to said patient a pharmaceutical or liquid composition according to any one of the preceding claims.

103. 103. The method of claim 102, wherein the proliferative disorder is cancer.

104. 104. The method of claim 103, wherein the cancer is a B cell proliferative disorder.

105. 105. The method of claim 104, wherein the B-cell proliferative disorder is selected from the group consisting of lymphoma, myeloma, non-Hodgkin's 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 lymphocytic leukemia (ALL), and mantle cell lymphoma.

106. 106. The method of claim 105, wherein the B cell proliferative disorder is non-Hodgkin's lymphoma (NHL).

107. 106. The method of claim 105, wherein the B cell proliferative disorder is diffuse large B cell lymphoma (DLBCL).

108. 106. The method of claim 105, wherein the B cell proliferative disorder is relapsed or refractory NHL.

109. 106. The method of claim 105, wherein the B cell proliferative disorder is follicular lymphoma (FL).

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