RU2800803C2 - STABLE COMPOSITIONS OF IMMUNOCONJUGATES CONTAINING ANTIBODIES TO CD79b - Google Patents

Abstract

FIELD: pharmaceuticals; medicine.

SUBSTANCE: object 1 is a stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, containing an immunoconjugate, a buffering agent, sugar and a surfactant. Objects 2–4 are a stable lyophilized composition for the introduction of an immunoconjugate containing an antibody to CD79b, containing an immunoconjugate, polysorbate 20, succinic acid, sodium hydroxide and sucrose. Objects 4–7 are a stable liquid composition for administration, in particular intravenous, immunoconjugate containing an antibody to CD79b. Objects 8–10 are the use of the stable composition in the manufacture of a medicinal product for the treatment of a B-cell proliferative disorder in a patient.

EFFECT: stability of immunoconjugates containing an antibody to CD79b.

37 cl, 18 dwg, 7 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. provisional application serial number 62/657185, filed April 13, 2018, which is incorporated herein by reference in its entirety.

REPRESENTATION OF A SEQUENCE LIST IN A TEXT FILE IN ASCII FORMAT

[0002] The contents of the following representation in an ASCII text file are incorporated herein in their entirety by reference: Computer Readable Form (CRF) of the Sequence List (File Name: 146392044340SEQLIST.TXT, Recorded on April 9, 2019, Size: 12 KB).

FIELD OF TECHNOLOGY

[0003] The present invention provides stable pharmaceutical compositions comprising an immunoconjugate containing an anti-CD79b antibody and a surfactant. The present invention also provides methods of using such compositions for the treatment of cancer.

BACKGROUND OF THE INVENTION

[0004] Intravenous (IV) administration using infusion bags is the most common way to deliver biologics commercially. To support the delivery and compatibility of a biologic, such as an immunoconjugate, when administered intravenously, it is necessary to develop a suitable therapeutic composition that remains stable after reconstitution in the infusion bag, during transport and throughout the course of administration (Bardin, C. et al. Annales pharmaceutiques francaises 69 (2011) 221-231).

[0005] One problem with the administration of biologics, especially with IV infusion bags, is that the materials of construction and the infusion solution can create a destabilizing environment for the therapeutic protein. In addition, the protein also encounters interfacial stresses in the IV bag. Protein adsorption at solid-liquid and air-water interfaces can cause protein denaturation at the surface leading to protein aggregation (Shieh, I., et al. Mol Pharm. 12 (2015): 3184-93; Sreedhara, A., et al Pharm Sci 101 (2012): 21-30). Further complicating IV administration, shaking infusion bags causes a constant regeneration of the air-liquid interface, resulting in repeated protein damage over time. Aggregation caused either by the destabilizing conditions of the IV bag solution or by interfacial stress can significantly and adversely affect product quality, efficacy, and biologic immunogenicity.

[0006] The deleterious effect of intravenous administration on biologics can be alleviated in part by the use of surfactants in formulation development. Non-ionic surfactants such as polysorbate-20 (PS20) or polysorbate-80 (PS80) are commonly used to protect and stabilize the molecule from air-water interfacial stress in protein preparations (Kerwin, BA. J. Pharm. Sci. 97 ( 2008) 2924-2935). Surfactants can protect the drug from surface damage by competing with protein at 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 critical disadvantage of using a nonionic surfactant at a concentration suitable for delivery in IV bags is that prolonged exposure of the biologic to the surfactant can result in the oxidation of certain amino acid residues, thereby reducing therapeutic efficacy. Lam X, et al., Pharm Res. (2011) 28:2543-2555.

[0007] It is important to note that prior to intravenous administration, biologics must also have a long stable shelf life under storage conditions, not including the structure and activity of the therapeutic protein. Liquid compositions of antibody-drug/drug conjugates (KALP) using a linker, for example, may be susceptible to acid-catalyzed hydrolysis of the linker during storage. Such instability can cause premature release of the drug when administered intravenously to a patient, adversely affecting the pharmacokinetics and safety of the biological product.

[0008] Thus, there is a need to develop a stable pharmaceutical composition that is both stable for intravenous administration and has a long shelf life under storage conditions. The present invention satisfies this need and offers other related advantages.

SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention provides a pharmaceutical composition comprising an immunoconjugate comprising an anti-CD79b antibody and a surfactant, wherein the concentration of the surfactant is at least 0.06% w/v. (for example, 0.6 mg/ml), and while the immunoconjugate containing the antibody to CD79b has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, while the light chain contains

(a) the HVR-L1 sequence KASQSVDYEGDSFLN (SEQ ID NO: 1);

(b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); And

(c) HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4);

(b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is from about 1 to about 8 (eg, from about 2 to about 5, eg, about 3.5).

[0010] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate is from about 5 mg/mL to about 60 mg/mL, from about 10 mg/mL to about 50 mg/mL, from 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 concentration of the anti-CD79b antibody immunoconjugate is from about 10 mg/mL to about 20 mg/mL. In one embodiment, the concentration of the anti-CD79b immunoconjugate is about 10 mg/mL. In another embodiment, the concentration of the anti-CD79b immunoconjugate is about 20 mg/mL.

[0012] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate is 5 mg/mL to 60 mg/mL, 10 mg/mL to 50 mg/mL, 10 mg/mL to 40 mg/mL, 10 mg/ml to 30 mg/ml or 10 mg/ml to 20 mg/ml.

[0013] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate is between 10 mg/mL and 20 mg/mL. In one embodiment, the concentration of the anti-CD79b immunoconjugate is 10 mg/mL. In another embodiment, the concentration of the anti-CD79b immunoconjugate is 20 mg/mL.

[0014] In some embodiments, the concentration of surfactant is from about 0.06% wt./about. (i.e. 0.6 mg/ml) to about 0.12% wt./about. (i.e. 1.2 mg/ml). In some embodiments, the concentration of surfactant is at least about 0.06% w/v. (i.e. 0.6 mg/ml). In another implementation, the concentration of surfactant is at least about 0.12% wt./about. (i.e. 1.2 mg/ml). In some embodiments, the concentration of surfactant is at least 0.06% w/v. In another implementation, the concentration of surfactant is at least 0.12% wt./about. In some embodiments, the concentration of surfactant is 0.06% w/v. In another implementation, the concentration of surfactant is 0.12% wt./about.

[0015] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate is about 10 mg/mL and the surfactant concentration is about 0.06% w/v. (i.e. 0.6 mg/ml). In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody is about 20 mg/mL and the concentration of the surfactant is at least about 0.12% w/v. In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody is about 20 mg/mL and the concentration of the surfactant is about 0.12% w/v. (i.e. 1.2 mg/ml).

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

[0017] In some embodiments according to (or with respect 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 at least for about 24, 48 or 72 hours when stored at 2-8°C.

[0018] In some embodiments of this invention, 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 particular embodiment, the surfactant is PS20. In yet another specific embodiment, the surfactant is PS80.

[0019] In some embodiments of this invention, the composition further comprises a buffering agent. In some embodiments, the buffering agent is a histidine buffer. In some embodiments, the buffering agent is a succinate buffer. In some embodiments, the succinate buffer is sodium succinate buffer. In some embodiments, the concentration of sodium succinate buffer is from about 10 mM to about 200 mM. In some embodiments, the concentration of sodium succinate buffer is between 10 mM and 200 mM. In one embodiment, the concentration of sodium succinate buffer is about 10 mM. In another embodiment, the concentration of sodium succinate buffer is 10 mM.

[0020] In some embodiments, the composition of this invention has a pH of about 5.0 to about 6.0. In particular 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 implementation of the composition in accordance with this invention has a pH of about 5.3. In some embodiments, the composition in accordance with this invention has a pH of 5.0 to 6.0. In 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 . In one embodiment, the implementation of the composition in accordance with this invention has a pH of 5.3.

[0021] In some embodiments of this invention, the composition further comprises sugar. In some embodiments, the sugar concentration is from about 100 mM to about 260 mM. In some embodiments, the sugar concentration is between 100 mM and 260 mM. In some embodiments, the sugar is selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, dextran 40, and trehalose.

[0022] In a particular embodiment, the sugar is sucrose. In one embodiment of this invention, the concentration of sucrose is about 120 mm. In another implementation, the concentration of sucrose is 120 mm.

[0023] In some embodiments, the composition of this invention is lyophilized (eg, as a lyophilized mass). In some embodiments, the lyophilized composition is contained in a vial, such as a 20 ml glass vial.

[0024] In one aspect, this invention provides a pharmaceutical composition obtained by lyophilizing a liquid composition containing 20 mg/ml of an immunoconjugate containing an anti-CD79b antibody in 10 mM sodium succinate buffer, 0.12% w/v. polysorbate 20 and 120 mm sucrose, and the liquid composition has a pH of 5.3, and the immunoconjugate containing the antibody to CD79b has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, wherein the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5).

[0025] In some embodiments of the invention, 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. B In some embodiments of this invention, the heavy chain contains the amino acid sequence of SEQ ID NO: 9, and the light chain contains the amino acid sequence of SEQ ID NO: 10.

[0026] Also provided herein is a pharmaceutical composition obtained by lyophilization of a liquid composition containing 20 mg/ml of an immunoconjugate containing an anti-CD79b antibody in 10 mM sodium succinate buffer, 0.12% w/v. polysorbate 20 and 120 mm sucrose, and the liquid composition has a pH of 5.3, and the immunoconjugate containing the antibody to CD79b has the formula:

where Ab is an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the heavy chain contains the amino acid sequence of SEQ ID NO: 9, the light chain contains the amino acid sequence of SEQ ID NO: 10; Val is valine; Cit is citrulline; and p is from about 2 to about 5 (eg, about 3.5). In some embodiments, the pharmaceutical composition is a lyophilized mass.

[0027] In some embodiments, the pharmaceutical composition of this invention is stable for about 60 months at 5°C±3°C protected from light. In a certain implementation, the pharmaceutical composition in accordance with this invention is stable for about 48 months at 5°C±3°C protected from light.

[0028] In some embodiments, the stability of a pharmaceutical composition in accordance with this invention is measured using size exclusion high performance liquid chromatography (E-HPLC). In one embodiment, the composition has a main peak (area %) of at least 95.0 as measured by e-HPLC.

[0029] In some embodiments, the stability of a pharmaceutical composition in accordance with this invention is measured using image-controlled capillary isoelectric focusing (ICEF). In one embodiment, the composition has a major peak (area %) of at least 58.0, an acidic region (area %) of at most 32.0, and a major region (area %) of at most 12.0, as measured by ucIEF.

[0030] In some embodiments, the pharmaceutical composition of the invention is reconstituted with sterile water for injection (SWI). In some embodiments, the pharmaceutical composition is reconstituted in about 7.2 ml of SVDI. 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 may be further diluted with isotonic buffer in an intravenous (IV) sachet. In some embodiments, the final volume of the diluted composition in the IV bag is from about 50 ml to about 100 ml. In some embodiments, the concentration of the immunoconjugate in the IV bag is from about 0.72 mg to about 2.7 mg.

[0031] Also provided herein is a pharmaceutical composition obtained by lyophilization of a liquid composition containing 20 mg/ml of an immunoconjugate containing an anti-CD79b antibody in 10 mM sodium succinate buffer, 0.12% w/v. polysorbate 20 and 120 mm sucrose, and the liquid composition has a pH of 5.3, and the immunoconjugate containing the antibody to CD79b has the formula:

where Ab is an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the heavy chain contains the amino acid sequence of SEQ ID NO: 9, the light chain contains the amino acid sequence of SEQ ID NO: 10; Val is valine; Cit is citrulline; and p is from about 2 to about 5 (eg, about 3.5).

[0032] A liquid formulation (eg, for intravenous administration) is provided, comprising a) about 0.72 to about 2.7 mg/ml of polatuzumab vedotin; b) about 0.36 mM to about 1.35 mM sodium succinate; c) about 0.51 mM to about 16.24 mM sucrose; d) from about 0.0432 mg/ml to about 0.162 mg/ml of polysorbate 20, wherein the pH of the liquid composition is from about 5 to about 5.7. Also provided is a liquid composition (eg, for intravenous administration) containing a) about 0.72 mg/ml of polatuzumab vedotin; b) about 0.36 mM sodium succinate; c) about 0.51 mM sucrose; d) about 0.0432 mg/ml polysorbate 20, wherein the pH of the liquid composition is from about 5.1 to about 5.4. Also provided is a liquid composition (eg, for intravenous administration) containing a) about 2.7 mg/ml of polatuzumab vedotin; b) about 1.35 mM sodium succinate; c) about 16.24 mM sucrose; d) about 0.162 mg/ml polysorbate 20, wherein the pH of the liquid composition is from about 5.1 to about 5.4. In some embodiments, the volume of the liquid composition is from 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) sachet. In some embodiments, the surfaces of the IV bag that contact the liquid composition are polyvinyl chloride (PVC), polyolefin (PO), polyethylene (PE), or polypropylene (PP).

[0033] In one aspect, the present invention provides a pharmaceutical composition comprising an immunoconjugate comprising an anti-CD79b antibody, a surfactant, a succinate buffer and a sugar, wherein the pharmaceutical composition, when reconstituted in water, forms a liquid pharmaceutical composition comprising an immunoconjugate comprising an anti-CD79b antibody. , at a concentration of from about 10 mg/kg to about 20 mg/ml, surfactant at a concentration of at least 0.06% wt./about. (i.e., 0.6 mg/mL), succinate buffer at a concentration of about 10 mM to about 200 mM, and sugar at a concentration of about 100 mM to about 260 mM, wherein the liquid pharmaceutical composition has a pH of 5.3, and at This immunoconjugate containing the antibody to CD79b has the formula:

where Ab is an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5). In some embodiments, 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, heavy the anti-CD79b antibody chain contains the amino acid sequence of SEQ ID NO: 9, while the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 10.

[0034] In some embodiments, the pharmaceutical composition is reconstituted in CVID and then diluted with buffer in an IV bag. In some embodiments, the pharmaceutical composition is reconstituted in CVID and then diluted with isotonic buffer in an IV bag. In one embodiment, the concentration of the surfactant when diluted in the IV bag is at least 0.003% w/v. In one embodiment, the concentration of the surfactant when diluted in the IV bag is at least 0.004% w/v. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In a particular embodiment, the sucrose concentration is 120 mM. In some embodiments, the succinate buffer is sodium succinate buffer. In a specific embodiment, the concentration of sodium succinate buffer is 10 mM. In some embodiments, the pharmaceutical composition after reconstitution 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 pharmaceutical composition, after reconstitution, is stable for at least 24, 48, or 72 hours when stored at about 2°C to about 8°C.

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

[0036] In certain implementations, the stability of the composition is measured using image-controlled capillary isoelectric focusing (ICEF). In a specific embodiment, the composition has a major peak (area %) of at least 58.0, an acidic region (area %) of at most 32.0, and a major region (area %) of at most 12.0, as measured by the ucIEF.

[0037] In some embodiments, the pharmaceutical composition described herein is contained in a glass vial (eg, a 20 ml glass vial). In some embodiments, the pharmaceutical composition is a lyophilized pharmaceutical composition. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition.

[0038] In one aspect, this invention provides a pharmaceutical composition obtained by a method comprising the steps of: (a) lyophilizing a liquid composition containing 20 mg/ml of an immunoconjugate containing an anti-CD79b antibody in 10 mM sodium succinate buffer, 0.12 wt. ./about. polysorbate 20 and 120 mm sucrose, and the liquid composition has a pH of 5.3, and the immunoconjugate containing the antibody to CD79b has the formula:

where Ab is an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the heavy chain contains the amino acid sequence of SEQ ID NO: 9, the light chain contains the amino acid sequence of SEQ ID NO: 10; Val is valine; Cit is citrulline; and p is from about 2 to about 5, to obtain a lyophilized composition; (b) reconstituting the lyophilized composition with about 7.2 ml of sterile water for injection (SWI) to obtain a reconstituted composition; and (c) diluting the reconstituted composition with isotonic buffer in an intravenous (IV) bag to obtain a pharmaceutical composition, wherein the final volume of the pharmaceutical composition in the IV bag is about 100 ml, and the final concentration of the immunoconjugate in the pharmaceutical composition is about 0.72 mg/ml or about 2.7 mg/ml.

[0039] In one aspect, the invention provides a liquid composition comprising an immunoconjugate comprising an anti-CD79b antibody, a surfactant, a succinate buffer, and a sugar, wherein the immunoconjugate comprising the anti-CD79b antibody 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% wt./about. (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, with the liquid composition having a pH of 5.3, and at This immunoconjugate containing the antibody to CD79b has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, wherein the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is from about 1 to about 8 (eg, from about 2 to about 5, eg, about 3.5). In some embodiments, 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, heavy the anti-CD79b antibody chain contains the amino acid sequence of SEQ ID NO: 9, while the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 10.

[0040] In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In a specific implementation, the concentration of sucrose is 120 mm. In some embodiments, the succinate buffer is sodium succinate buffer. In a specific embodiment, the concentration of sodium succinate buffer is 10 mM.

[0041] In some embodiments, the liquid composition in accordance with this invention is diluted with isotonic buffer. In an exemplary embodiment, the liquid composition according to the present invention, dissolved in isotonic buffer, is in an IV bag. In some embodiments, the isotonic buffer is 0.9% sodium chloride solution, 0.45% sodium chloride solution, or 5% dextrose solution.

[0042] In some embodiments, the liquid composition provided herein, dissolved in 0.9% sodium chloride solution, is stable (according to any one or more of the criteria described elsewhere herein) after dilution for at least about 24 hours at 2-8°C. In some embodiments, the liquid composition provided herein, when dissolved in 0.9% sodium chloride solution, is stable (by any one or more of the criteria described elsewhere herein) after dilution for up to about 4 hours at a temperature from 9°С to 25°С. In some embodiments, the liquid composition provided herein, dissolved in 0.45% sodium chloride solution, is stable (according to any one or more of the criteria described elsewhere herein) after dilution for at least about 24 hours at 2°C-8°C. In some embodiments, the liquid composition provided herein, dissolved in 0.45% sodium chloride solution, is stable (according to any one or more of the criteria described elsewhere herein) after dilution for about 4 hours at a temperature of 9°C to 25°C. In some embodiments, the liquid composition provided herein, when dissolved in a 5% dextrose solution, is stable (according to any one or more of the criteria described elsewhere herein) after dilution for at least about 48 hours at 2°C. C-8°C. In some embodiments, the liquid composition provided herein, when dissolved in a 5% dextrose solution, is stable (according to any one or more of the criteria described elsewhere herein) after dilution for about 8 hours at 9°C. up to 25°С.

[0043] In some embodiments, the liquid composition dissolved in isotonic buffer is in an IV bag. In some embodiments, the surfaces of the IV bag that contact the composition that is dissolved in the isotonic buffer consist of polyvinyl chloride (PVC), polyolefin (PO), polyethylene (PE), or polypropylene (PP).

[0044] In some embodiments, the liquid composition of this invention, when dissolved in isotonic buffer in an IV bag, is stable for up to about 6 to about 8 hours at 30°C. In some embodiments, the liquid composition of this invention, dissolved in isotonic buffer in an IV bag, is stable for about 24 hours at 25°C. In some embodiments, the liquid composition of this invention, when dissolved in isotonic buffer in an IV bag, is stable for up to about 72 hours at 5°C±3°C. In some embodiments, the isotonic buffer is 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.

[0045] A lyophilized pharmaceutical composition is also provided, containing about 150 mg of an immunoconjugate containing an anti-CD79b antibody, 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, and an immunoconjugate containing an anti-CD79b antibody has the formula:

where Ab is an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the heavy chain of the antibody to CD79b contains the amino acid sequence of SEQ ID NO: 9, and the light chain of the antibody to CD79b contains the amino acid sequence of SEQ ID NO : 10; Val is valine; Cit is citrulline; and p is from about 2 to about 5 (eg, about 3.5). In some embodiments, the lyophilized composition (eg, lyophilized mass) is contained in a vial, eg, a 20 ml glass vial.

[0046] In addition, a lyophilized pharmaceutical composition is provided containing about 150 mg of polatuzumab vedotin, about 9.0 mg of polysorbate 20, about 8.88 mg of succinic acid, about 4.08 mg of sodium hydroxide, and about 309 mg of sucrose. In some embodiments, the lyophilized composition (eg, lyophilized mass) is contained in a vial, eg, a 20 ml glass vial.

[0047] Also provided is 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, moreover, the immunoconjugate containing the antibody to CD79b has the formula:

where b represents an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the heavy chain of the antibody to CD79b contains the amino acid sequence of SEQ ID NO: 9, and the light chain of the antibody to CD79b contains the amino acid sequence of SEQ ID NO : 10; Val is valine; Cit is citrulline; and p is from about 2 to about 5 (eg, about 3.5). In some embodiments, the lyophilized composition (eg, lyophilized mass) is contained in a vial, eg, a 20 ml glass vial.

[0048] In addition, a lyophilized pharmaceutical composition is provided containing about 140 mg of polatuzumab vedotin, about 8.4 mg of polysorbate 20, about 8.27 mg of succinic acid, about 3.80 mg of sodium hydroxide, and about 288 mg of sucrose. In some embodiments, the lyophilized composition (eg, lyophilized mass) is contained in a vial, eg, a 20 ml glass vial.

[0049] Also provided is a lyophilized pharmaceutical composition containing about 30 mg of an anti-CD79b antibody immunoconjugate, about 1.8 mg of polysorbate 20, about 1.77 mg of succinic acid, about 0.816 sodium hydroxide, and about 61.8 mg of sucrose, wherein an immunoconjugate containing an anti-CD79b antibody has the formula:

where Ab is an antibody to CD79b, while the antibody to CD79b contains a heavy chain and a light chain, while the heavy chain of the antibody to CD79b contains the amino acid sequence of SEQ ID NO: 9, and the light chain of the antibody to CD79b contains the amino acid sequence of SEQ ID NO : 10; Val is valine; Cit is citrulline; and p is from about 2 to about 5 (eg, about 3.5). In some embodiments, the lyophilized composition is a lyophilized mass. In some embodiments, the lyophilized composition (eg, lyophilized mass) is contained in a vial, eg, a 20 ml glass vial.

[0050] In addition, a lyophilized pharmaceutical composition is provided containing about 30 mg of polatuzumab vedotin, about 1.8 mg of polysorbate 20, about 1.77 mg of succinic acid, about 0.816 mg of sodium hydroxide, and about 61.8 mg of sucrose. In some embodiments, the lyophilized composition (eg, lyophilized mass) is contained in a vial, eg, a 20 ml glass vial.

[0051] In some embodiments, the lyophilized pharmaceutical composition provided herein is a lyophilized mass. In some embodiments, a lyophilized pharmaceutical composition according to any of the embodiments of this document is stable for at least 6, 12, 18, 24, 30, 36, 42, 48, 54, or 60 months when stored at a temperature of from about 2°C. up to about 8°C.

[0052] A pharmaceutical composition is provided comprising a) 5-60 mg/ml of Polatuzumab vedotin; b) 10-200 mM sodium succinate; c) 100-260 mM sucrose; and d) 0.06-0.12% polysorbate 20, wherein the liquid composition has a pH of 5 to 6. In some embodiments, the liquid pharmaceutical composition contains a) 10-55 mg/ml of polatuzumab vedotin;

b) 10-100 mM sodium succinate; c) 150-260 mM sucrose; and d) 0.08-0.12% w/v. polysorbate 20, while the pH of the liquid composition is from 5.1 to 5.6. In some embodiments, the liquid pharmaceutical composition contains a) 15-40 mg/ml of polatuzumab vedotin; b) 10-50 mM sodium succinate;

c) 200-260 mM sucrose; and d) 0.1-0.12% w/v. polysorbate 20, while the pH of the liquid composition is from 5.2 to 5.4. In some embodiments, a liquid composition is obtained by reconstituting a lyophilized composition (such as a lyophilized mass). In some embodiments, the liquid composition is contained in a vial, such as a 20 ml glass vial.

[0053] Also provided is a liquid pharmaceutical composition comprising a) 20 mg/mL of Polatuzumab vedotin; b) 10 mM sodium succinate; c) 120 mM sucrose; and d) 0.12% w/v. polysorbate 20, while the pH of the liquid composition is about 5.3. In some embodiments, a liquid composition is obtained by reconstituting a lyophilized composition (such as a lyophilized mass). In some embodiments, the liquid composition is contained in a vial, such as a 20 ml glass vial.

[0054] In some embodiments, the liquid composition of any of the embodiments provided herein is stable for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours at storage at about 30°C. In some embodiments, the liquid composition of any of the embodiments provided herein is stable for at least 24, 48, or 72 hours when stored at about 2°C to about 8°C. In some embodiments, a liquid composition is obtained by reconstituting a lyophilized composition (eg, a lyophilized mass). In some embodiments, the liquid composition is contained in a vial, such as a 20 ml glass vial.

[0055] In some embodiments, the reconstituted pharmaceutical composition provided herein is stable after 72 hours of storage at 2°C-8°C. In some embodiments, the reconstituted pharmaceutical composition provided herein is stable after 24 hours of storage at 30°C when exposed to ambient light. In some embodiments, the reconstituted pharmaceutical composition contains 20 mg/ml of polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, and 1.2 mg/ml polysorbate 20, with a pH of 5.3. In some embodiments, the affinity of polatuzumab vedotin for its target (ie, CD79b) or for the biological activity of polatuzumab vedotin.

[0056] In one aspect, this invention provides a method of treating a proliferative disorder in a patient in need thereof, comprising administering to the patient a pharmaceutical composition or liquid composition described herein. Also provided is the use of the pharmaceutical composition or liquid composition described herein for the manufacture of a medicament for the treatment of a proliferative disorder in a patient in need thereof. In some embodiments, a pharmaceutical composition or liquid composition described herein is provided for use in the treatment of a proliferative disorder in a patient in need thereof. Also provided is a pharmaceutical composition or liquid composition described herein for use in a method for treating a proliferative disorder in a patient in need thereof.

[0057] In one embodiment, the proliferative disorder is a malignancy. In an exemplary embodiment, the cancer is a disorder of B cell proliferation. In specific embodiments, the disorder of B cell proliferation is selected from the group consisting of: lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), relapsed/refractory forms of 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 cell lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0058] In one embodiment of the invention, the B cell proliferation disorder is non-Hodgkin's lymphoma (NHL). In one embodiment of the present invention, the B cell proliferation disorder is diffuse large B cell lymphoma (DLBCL). In one embodiment of the invention, the B cell proliferation disorder is relapsing/refractory DLBCL. In some embodiments of the invention, the B cell proliferation disorder is relapsing/refractory DLBCL. In another embodiment of the present invention, the B cell proliferation disorder is relapsing NHL or refractory NHL. In yet another embodiment of the invention, the B cell proliferation disorder is follicular lymphoma (FL).

BRIEF DESCRIPTION OF GRAPHICS

[0059] In FIG. 1 shows the increase in macromolecular compounds (HMCs) after 22 hours of static storage at 30°C with a fixed concentration of PS20 ([PS20]) in three different sizes of bags for intravenous (IV) administration.

[0060] In FIG. 2 shows the increase in the amount of IUD after 2 hours of mixing (100 rpm) at 30° C. with a fixed concentration of PS20 ([PS20]) in three different sizes of IV bags.

[0061] In FIG. 3 illustrates the effect of temperature on the physical stability of anti-CD79b-vc-MMAE in IV bags with saline at different concentrations of PS20 ([PS20]).

[0062] In FIG. 4 illustrates the effect of surfactant type and infusion fluid during static storage at 30° C. for 22 hours.

[0063] In FIG. 5A illustrates the effect of 2-8°C temperature on the stability of anti-CD79b-vc-MMAE under agitation. On FIG. 5B illustrates the effect of 25° C. on the stability of anti-CD79b-vc-MMAE under agitation. On FIG. 5C illustrates the effect of 30° C. on the stability of anti-CD79b-vc-MMAE under agitation.

[0064] In FIG. 6 illustrates the physical stability of anti-CD79b-vc-MMAE using mixing model 1.

[0065] In FIG. 7 illustrates the physical stability of anti-CD79b-vc-MMAE using mixing model 2.

[0066] In FIG. 8 illustrates a thiosuccinimide hydrolysis reaction to stabilize the immunoconjugate and prevent elimination. Maleimide elimination can be prevented by hydrolysis of the thiosuccinimide bonds present in the immunoconjugate.

[0067] In FIG. 9 illustrates the effect of pH on acid charge variant formation at 30° C. at three different time points: 2 weeks, 4 weeks, and 8 weeks.

[0068] In FIG. 10 illustrates the effect of pH on base charge variant formation at 30° C. at three different time points: 2 weeks, 4 weeks, and 8 weeks.

[0069] In FIG. 11 illustrates the effect of pH on IUD formation at 30° C. at three different time points: 2 weeks, 4 weeks and 8 weeks.

[0070] In FIG. 12 illustrates the effect of pH and buffer species on small molecule compounds (LMCs) stressed at 30° C. for 4 weeks.

[0071] In FIG. 13 illustrates the stability of freeze-dried compositions at 30° C. as measured by size exclusion chromatography (SEC).

[0072] In FIG. 14 illustrates the stability of lyophilized formulations at 30° C. measured using image-controlled capillary isoelectric focusing (ICEFv).

[0073] In FIG. 15A-15C illustrate the appearance of a lyophilized mass with various ratios of protein to sucrose. Fig. 15A represents 10 mg/ml anti-CD79b-vc-MMAE and 260 mM sucrose. Fig. 15B represents 10 mg/ml anti-CD79b-vc-MMAE and 180 mM sucrose. Fig. 15C represents 10 mg/ml anti-CD79b-vc-MMAE and 120 mM sucrose.

[0074] In FIG. 16A-16B illustrate the appearance of freeze-dried mass with different ratios of protein to sucrose. Fig. 16A represents 10 mg/ml anti-CD79b-vc-MMAE and 260 mM sucrose. Fig. 16B represents 20 mg/ml anti-CD79b-vc-MMAE and 120 mM sucrose.

[0075] In FIG. 17 illustrates the stability of lyophilized anti-CD79b-vc-MMAE drug stressed at 2-8°C, 25°C, and 40°C for measurement of IUD variants by SEC.

[0076] In FIG. 18 illustrates the stability of lyophilized anti-CD79b-vc-MMAE drug stressed at 2-8°C, 25°C, and 40°C to measure IUD variants by vcIEF.

DETAILED DESCRIPTION OF THE INVENTION

[0077] This invention provides stable pharmaceutical compositions containing an immunoconjugate containing an antibody to CD79b, and a surfactant. The present invention also provides methods of using such compositions for the treatment of cancer.

I. Definitions

[0078] It should be understood that this description is not limited to specific compositions or biological systems, which, of course, may vary. It should also be understood that the terminology used herein is only intended to describe specific implementations and is not intended to be limiting. In the context of this description and the appended claims, the singular forms in the context of this document include the plural forms, unless the content clearly dictates otherwise. Thus, for example, reference to "molecule" does not necessarily include a combination of two or more such molecules, and the like.

[0079] The term "about" as used herein refers to the typical error range for the corresponding value and is well known to one of skill in the art. As used herein, the use of "about" in relation to a value or parameter includes (and describes) implementations that refer directly to that value or parameter.

[0080] It is to be understood that the aspects and embodiments of the present invention described herein include "comprising", "consisting of", and "consisting essentially of" the aspects and embodiments of the present invention.

[0081] The term "anti-CD79b antibody-containing immunoconjugate" refers to an anti-CB79b antibody-drug/drug (KALP) conjugate. As used herein, an anti-CD79b immunoconjugate comprises an antibody or fragment thereof capable of binding CD79b, a linker, and a drug molecule. The term "linker", as used herein, refers to 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB).

[0082] The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-CD79b monoclonal antibodies (including, without limitation, agonistic, antagonistic, neutralizing antibodies, full-length or intact monoclonal antibodies), compositions of anti-CD79b antibodies with polyepitope specificity , polyclonal antibodies, polyvalent antibodies, polyspecific antibodies (for example, bispecific antibodies if they exhibit the desired biological activity) formed from at least two intact antibodies, single-chain antibodies to CD79b and fragments of antibodies to CD79b (see below), including Fab, Fab', F(ab')2 and Fv fragments, diabodies, single domain antibodies (sdAb), provided they exhibit the desired biological or immunological activity. The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein. The antibody can be a chimeric, human, humanized, and/or affinity matured antibody.

[0083] The term "anti-CD79b antibody" or "CD79b-binding antibody" refers to an antibody that is capable of binding CD79b with sufficient affinity such that the antibody is suitable as a diagnostic and/or therapeutic agent when targeting CD79b. Preferably, the degree of binding of an anti-CD79b antibody to an unrelated, non-CD79b protein is less than about 10% of the binding of the antibody to CD79b as measured, for example, by radioimmunoassay (RIA). In some 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 a CD79b epitope that is conserved among CD79b from different species.

[0084] The 4-chain antibody core unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 basic heterotetrameric units along with an additional polypeptide called the J chain , and therefore contain 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form polyvalent systems containing 2-5 4-strand base units along with the J-chain). In the case of IgG, a 4-chain element typically has a molecular weight of about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain. Each H and L chain also has equally 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 ji and e isotypes. Each L-chain has at the N-terminus a variable domain (VL) followed by a constant domain (CL) at the other end. VL is aligned with VH and CL is aligned with the first heavy chain constant domain (CH1). It is believed that specific amino acid residues form the contact surface between the variable domains of the light and heavy chains. Pairing VH and VL forms a single antigen-binding site. For information on the structure and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0085] The L chain of any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of their heavy chain constant domain (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, β, ε, γ, and μ, respectively. The γ and α classes are further subclassified based on relatively minor differences in CH sequence and function, for example, the following subclasses are expressed in humans: IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0086] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an 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 usually the most variable parts of an antibody and contain antigen binding sites.

[0087] The term "variable" refers to the fact that the sequences of certain segments of the variable domains differ significantly among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not uniform across a stretch of 110 amino acid variable domains. Instead, V regions are composed of relatively invariant regions called framework regions (FRs) of 15-30 amino acids separated by shorter regions of high variability called "hypervariable regions" that are 9-12 amino acids long. Each native light and heavy chain variable domain contains four FR regions, predominantly assuming a β-layer configuration, connected by three hypervariable regions that form loops that unite the β-layer structure, and, in some cases, form part of it. The hypervariable regions in each chain are located together in close proximity to the FR and, together with the hypervariable regions of the other chain, are involved in the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, M.D. (1991)). The constant domains are not directly involved in antibody-antigen binding, but they exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0088] An "intact" antibody is an antibody that contains an antigen-binding site, as well as CL and at least the heavy chain constant domains, CH1, CH2, and CH3. The constant domains can be native sequence constant domains (eg, human native sequence constant domains) or an amino acid sequence variant thereof. Preferably, the intact antibody performs one or more effector functions.

[0089] A "naked antibody" for the purposes of this disclosure is an antibody that is not conjugated to a drug moiety or radioactive label.

[0090] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabody; linear antibodies (see, for example, US patent No. 5641870, example 2, Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single chain antibody molecules; and polyspecific antibodies formed by antibody fragments. In one embodiment, the antibody fragment contains the antigen-binding site of an intact antibody and thus retains the ability to bind an antigen.

[0091] Cleavage of antibodies with papain results in the formation of two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to easily crystallize. The Fab fragment consists of the entire L chain along with the H chain variable region domain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent in terms of antigen binding, ie. it has one antigen-binding site. Treatment of the antibody with pepsin results in one large F(ab')2 fragment, which approximately corresponds to two disulfide-linked Fab fragments having bivalent antigen-binding activity and, in addition, is capable of antigen cross-linking. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl end of the CH1 domain, containing one or more cysteine residues from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains has a free thiol group. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments that have hinged cysteines between them. In addition, other variants of chemical conjugation of antibody fragments are known.

[0092] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by the sequences of the Fc region, which is also the part recognized by the Fc receptors (FcRs) found on certain cell types.

[0093] "Fv" is the minimum antibody fragment containing a complete antigen recognition and antigen binding site. This fragment consists of a dimer consisting of one heavy chain variable domain and one light chain variable domain linked by a strong non-covalent bond. In single chain Fv types (scFv), one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can form a "dimeric" structure similar to that of double chain Fv types. The folding of these two domains forms six hypervariable loops (3 loops in each of the H and L chains) that provide amino acid residues for antigen binding and provide specificity for antibody antigen binding. However, even a single variable domain (or half of the Fv containing only three antigen-specific HVRs) has the ability to recognize and bind an antigen, albeit at a lower affinity than the entire binding site.

[0094] "Single chain Fv", also abbreviated as "sFv" or "scFv", are antibody fragments that contain the VH and VL domains of an antibody joined into a single polypeptide chain. Preferably, the sFv polypeptide also optionally contains a polypeptide linker between the VH and VL domains that allows the formation of the sFv structure required for antigen binding. For a review of sFv fragments, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, below.

[0095] The term "diabodies" refers to antibody fragments containing two antigen-binding sites, said fragments containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) of the same polypeptide chain (VH-VL). Small antibody fragments are generated by constructing sFv fragments (see previous paragraph) with short linkers (approximately 5-10 residues) between the VH and VL domains such that interchain, but not intrachain, V-domain pairing is allowed, resulting in divalent fragment, i.e. a fragment with two antigen-binding sites. Diabodies can be divalent or bispecific. Bispecific diabodies are heterodimers of two "crossing" sFv fragments, in which the VH and VL domains of the two antibodies are located on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404097; 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). Triatebodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0096] The term "monoclonal antibody", in the context of this document, refers to an antibody from a collection of essentially homogeneous antibodies, i. the individual antibodies constituting a population are identical except for possible mutations, such as naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, 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 antibody preparations are advantageous in that they can be synthesized without contaminating other antibodies. The definition of "monoclonal" does not imply that the antibody must be obtained in any particular way. For example, monoclonal antibodies for use in accordance with the invention may be produced by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or they may be produced by recombinant DNA methods in bacterial, eukaryotic cells, and also in animal and plant cells (see, for example, US patent No. 4816567). "Monoclonal antibodies" can also be generated from antibody phage libraries using the methods described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0097] Monoclonal antibodies herein specifically include "chimeric" antibodies in which the heavy and/or light chain region is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remaining part of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see US patent No. 4816567; and Morrison et al., Proc Natl Acad Sci USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising non-human primate (eg, old world monkey, monkey, etc.) antigen-binding sequences and human constant domain sequences.

[0098] "Humanized" forms of antibodies of non-human origin (eg, rodents) are chimeric antibodies containing a minimum sequence derived from an antibody of non-human origin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primates , which have the desired antibody specificity, affinity and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve the parameters of the antibody. Typically, a humanized antibody contains substantially all of at least one, and typically two, variable domains wherein all or substantially all of the hypervariable loops correspond to non-human immunoglobulin hypervariable loops and all or substantially all of the FRs. α-regions are FR regions from a human immunoglobulin sequence. Also, the humanized antibody optionally comprises at least a portion of an immunoglobulin constant domain (Fc), typically a human immunoglobulin constant domain. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Biochem. soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0099] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody sequence produced in a human and/or obtained using any of the methods for producing human antibodies described herein. This definition of a human antibody specifically does not include a humanized antibody containing non-human antigen-binding residues. Human antibodies can be generated using a variety of methods known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are 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). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be obtained by administering an antigen to a transgenic animal modified to produce such antibodies in response to an antigen load, but having damaged endogenous loci, such as immunized xenomic mice (see, for example, US Pat. See also, for example, Li et al., Proc. Natl. Acad. sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B-cell hybridoma technology.

[0100] The term "hypervariable region", "HVR", or "HV", as used herein, refers to regions of the variable domain of an antibody that are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies include six hypervariable regions: three in VH (H1, H2, H3) and three in VL (LI, L2, L3). Use several ways to designate hypervariable regions, and they are included in this description. Complementarity-determining regions (CDRs) according to Kabat are based on sequence variability and are most commonly used by 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 arrangement of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the CDR-H1 no Chothia loop, when numbered using the Kabat numbering rule, varies between H32 and H34 depending on the length of the loop (this is because Kabatu's numbering scheme places inserts at H35A and H35B; if neither 35A, neither 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, then the loop ends at 34). AbM hypervariable regions are a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" hypervariable regions are based on the analysis of available complex crystal structures. Residues from each of these hypervariable regions are noted below.

[0101] Hypervariable regions may include the following "elongated hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 (L3) in VL and 26-35 B (H1), 50-65, 47-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) in VH. Variable domain residues are numbered according to Kabat et al., above for each of these definitions.

[0102] "Frame" residues or "FR" are variable domain residues other than hypervariable region residues as defined herein.

[0103] The terms "Kabat variable domain residue numbering" or "Kabat amino acid position numbering", and variants thereof, refer to the numbering system that is used for heavy chain variable domains or light chain variable domains in the antibody pool of Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the true linear amino acid sequence may contain fewer or more amino acids, corresponding to shortening or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may include one insertion of an amino acid of interest (Kabat residue 52a) after residue 52 in H2 and inserted residues (e.g., Kabat residues 82a, 82b, and 82c, etc.) after residue 82 FR heavy chain. Kabat residue numbering can be determined for a particular antibody by aligning the antibody sequence homology regions with a "standard" Kabat numbered sequence.

[0104] The Kabat numbering system is commonly used when referring to a residue in a variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (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 a residue in an immunoglobulin heavy chain constant region (eg, the EU index given in Kabat et al., supra). "Kabat's EU index" refers to the numbering of residues of the EU human IgG1 antibody. Unless otherwise indicated herein, references to residue numbers in the variable domains of antibodies mean the numbering of residues in accordance with the Kabatu numbering system. Unless otherwise indicated herein, references to residue numbers in the constant domain of antibodies mean residue numbering according to the EU numbering system (eg, see US Provisional Application No. 60/640323, EU Numbering Figures).

[0105] An antibody produced by "affinity maturation" is an antibody with one or more changes in one or more of its HVRs that result in an increase in the affinity of the antibody for the antigen compared to the parent antibody that does not have the change(s). Preferred antibodies produced by "affinity maturation" have nanomolar or even picomolar affinity for the target antigen. Affinity maturation antibodies are prepared by methods known in the art. In Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by shuffling the VH and VL domains. 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).

[0106] The term "binding affinity" generally refers to the strength of all overall non-covalent interactions between a single binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the methods described herein. Low affinity antibodies usually bind antigen slowly and tend to dissociate easily, while high affinity antibodies usually bind antigen faster and tend to stay bound longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this invention. Specific illustrative implementations are described below.

[0107] The expression "or better", as used herein, when referring to binding affinity, refers to a stronger binding between a molecule and its binding partner. "Or better", as used herein, refers to stronger binding having a lower Kd numerical value. For example, in the case of an antibody whose affinity for an antigen is ".6 nM or better", the affinity of the antibody for the antigen is <.6 nM, i.e., 59 nM, .58 nM, .57 nM, etc., or any a value that is less than 6 nM.

[0108] In one embodiment, the "Kd" or "Kd value" of the present invention is measured by a radiolabeled antigen (RIA) binding assay performed on a Fab version of an antibody of interest and its antigen, as described in the following assay , which measures the binding affinity of a Fab to an antigen in solution by equilibrating the Fab with a minimum concentration of (125I)-labeled antigen in the presence of a series of titers of unlabeled antigen, followed by fixation of the bound antigen on an anti-Fab coated plate (Chen, et al. (1999) J. Mol Biol 293:865-881). To establish assay conditions, microtiter plates (Dynex) were coated overnight with 5 μg/ml fixing antibody to Fab (Cappel Labs) in 50 mM sodium carbonate solution (pH 9.6) and then blocked using 2% (w/w) ./vol.) of bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-absorbent plate (Nunc #269620), 100 pM or 26 pM [125I] antigen are mixed with serial dilutions of the Fab of interest (e.g., according to the anti-VEGF antibody Fab-12, in Presta et al. (1997) Cancer Res. 57:4593-4599). The Fab of interest was then incubated overnight; however, incubation may be continued for a longer period (eg, about 65 hours) to ensure that equilibrium has been reached. Thereafter, the mixtures were transferred to a fixing plate for incubation at room temperature (eg, for 1 hour). The solution was then removed and the plate was washed eight times with 0.1% Tween-20 in PBS. After the plates are dry, 150 μl/well of scintillator (MicroScint-20; Packard) is added and the plates are counted using a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each of the Fabs that provide a binding less than or equal to 20% of the maximum binding are selected for use in competitive binding assays.

[0109] Alternatively, "Kd" or "Kd value" is measured using surface plasmon resonance analysis using a BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C. CM5 chips with antigen immobilized on them at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (~0.2 μM) prior to injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of bound protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. To determine the kinetics, two-fold serial dilutions of Fab (from 0.78 nM to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25°C with a flow rate of approximately 25 μl/min. The association constant (cop) and the dissociation constant ((koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously matching the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon See, for example, Chen, Y., et al (1999) J. Mol Biol 293:865-881 If the antibody association constant exceeds 106 M-1 s-1 in the assay by the surface plasmon resonance method indicated above, then the association constant can be determined using a fluorescence quenching method in which the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandwidth 16 nm) at 25° C. of 20 nM anti-antigen antibody (Fab form) is measured ) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured by a spectrophotometer such as a stop-jet spectrophotometer (Aviv Instruments) or an SLM-Aminco 8000 series spectrophotometer (Thermo Spectronic) with agitated cuvette.

[0110] "Binding rate" or "association rate" or "bonding rate" or "cop" according to this invention can also be determined using said surface plasmon resonance method described above using BIAcoreTM-2000 or BIAcoreTM-3000 ( BIAcore, Inc., Piscataway, NJ) as described above.

[0111] The expression "substantially similar" or "substantially the same", in the context of this document, means a sufficiently high degree of similarity between two numerical values (typically one associated with an antibody according to this invention, and the other associated with a reference/comparative antibody) so that one skilled in the art may consider the difference between the two values to be of little or no biological and/or statistical significance in the context of the biological trait measured by said values (eg, Kd values). The difference between these 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/comparative antibody .

[0112] The expression "significantly reduced" or "significantly different", in the context of this document, means a significant high degree of difference between two numerical values (typically one associated with an antibody according to this invention, and the other associated with a reference/comparative antibody), so that a person skilled in the art may consider the difference between the two values to be statistically significant in the context of the biological trait measured by said values (eg, Kd values, HAMA response). The difference between these two values is preferably greater than about 10%, preferably greater than about 20%, preferably greater than about 30%, preferably greater than about 40%, preferably greater than about 50% as a function of the value for the reference/comparative antibody .

[0113] For the purposes of this document, a "human acceptor framework" is a framework containing the amino acid sequence of a VL or VH framework formed by a human immunoglobulin framework or a human framework consensus sequence. The human acceptor framework "formed" by a human immunoglobulin framework or human framework consensus sequence may contain their amino acid sequence itself, or it may contain preexisting changes in amino acid sequences. Where pre-existing amino acid changes are present, it is preferred that no more than 5, and preferably 4 or less, or 3 or less, pre-existing amino acid changes be present. Where pre-existing amino acid changes in VH are present, it is preferred that these changes exist only at three, two, or one of the 71H, 73H, and 78H positions; for example, amino acid residues at these positions may be 71A, 73T and/or 78A. In one embodiment, the human VL acceptor framework is identical in sequence to the human immunoglobulin VL framework or to the consensus human framework sequence.

[0114] "Human framework consensus sequence" is a framework region that is the most frequently occurring amino acid residues when selecting human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Typically, a subgroup of sequences is a subgroup according to Kabat et al. In one embodiment, for VL, the subgroup is the kappa I subgroup of Kabat et al. In one embodiment, for VH, the subgroup is subgroup III of Kabat et al.

[0115] "Subgroup III VH consensus framework region" includes a consensus sequence formed by the amino acid sequences of the subgroup III heavy chain variable regions of Kabat et al. In one embodiment, the subgroup III VH consensus framework amino acid sequence includes at least some or all of each of the following sequences:

[0116] The “subgroup I VL consensus framework region” includes the consensus sequence formed by the amino acid sequences of the subgroup I kappa light chain variable regions of Kabat et al. In one embodiment, the amino acid sequence of the subgroup I VL consensus framework region includes at least some or all of each of the following sequences:

[0117] An "unmodified human framework" is a human framework that has the same amino acid sequence as the human acceptor framework, e.g., it lacks a human amino acid substitution(s) for a non-human amino acid in the human acceptor framework.

[0118] An anti-CD79b immunoconjugate or antibody that "binds" CD79b is one that binds the CD79b antigen with sufficient affinity such that the antibody is suitable as a drug to target to a cell or tissue expressing the antigen, and which does not cross-react with other proteins to a significant extent. In such embodiments, the degree of binding of the antibody to the "non-target" protein will be less than 10% of the binding of the antibody to its specific target protein, as determined by a fluorescence-activated cell sorting (FACS) assay or by radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds to" or is "specific to" a particular polypeptide or epitope on a particular target polypeptide means a binding that is at a measurable level different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of the molecule compared to the binding of a control molecule, which is usually a molecule with a similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated by the fact that binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. The terms "specific binding" or "specifically binds to" or is "specific to" a particular polypeptide or epitope on a particular target polypeptide, in the context of this document, can be illustrated, for example, by a molecule having a Kd for the target of at least about 10 4 M, alternatively at least about 10-5 M, alternatively at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10- 9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or more. In one embodiment, the term "specific binding" refers to binding in which an antibody binds to an epitope on a CD79b polypeptide without substantially binding to any other polypeptide or epitope of the polypeptide.

[0119] The "effector functions" of an antibody refer to those biological activities that are inherent in the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ACDD, ADCC); phagocytosis; down regulation of cell surface receptors (eg, B cell receptor; BCR) and activation of B cells.

[0120] As used herein, the term "Fc region" is used to refer to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as a stretch from the amino acid residue at position Cys226, or Pro230, to its carboxyl-terminus. The C-terminal lysine (residue 447 according to the EC numbering system) of the Fc region can be removed, for example, during the production or purification of an antibody, or during recombinant construction of a nucleic acid encoding an antibody heavy chain. Accordingly, an intact antibody composition may include antibody populations all of which have a K447 residue removed, antibody populations that have not had a K447 residue removed, and antibody populations containing a mixture of antibodies with and without a K447 residue.

[0121] A "functional Fc region" has an "effector function" of a native sequence Fc region. Examples of "effector functions" include C1q binding; KZTs; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (eg, B cell receptor; BCR), etc. Such effector functions typically require the Fc region to be combined with a binding domain (eg, an antibody variable domain) and can be assessed using various assays, as described, for example, in the "Definitions" section of this document.

[0122] A "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include, without limitation, 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.

[0123] A "variant Fc region" includes an amino acid sequence that differs from a native sequence Fc region in that it contains at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region comprises at least one amino acid substitution relative to the native sequence Fc region or the parent polypeptide Fc region, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions. substitutions in the Fc region with the native sequence or in the Fc region of the original polypeptide. As used herein, the Fc region variant preferably shares at least about 80% homology with the native sequence Fc region and/or Fc region of the parent polypeptide, and most preferably it shares at least about 90% homology with them, more preferably it shares at least about 95% homology with them.

[0124] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig associated with Fc receptors (FcRs) found on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages), cause specific binding for these cytotoxic effector cells to the antigen-bearing target cell, and then kill the target cells through cytotoxins. Antibodies are the "weapons" of cytotoxic cells and are absolutely necessary for such destruction. The main cells for ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is shown 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 can be performed, such as the assay described in US Pat. No. 5,500,362 or US Pat. NK). Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).

[0125] The terms "Fc receptor" or "FcR" refer to a receptor that

binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is an FcR 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 mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine inhibitory motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain. (See review by M in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are described 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). The term "FcR" in this document refers to other FcR, including FcR, which will be identified in the future. The term also includes the neonatal FcRn receptor which is responsible for 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)).

[0126] In vivo human FcRn binding and serum half-life for polypeptides with high human FcRn binding affinity can be analyzed, for example, in transgenic mice or in transfected human cell lines expressing human FcRn or in primates administered with variant polypeptides. Fc regions. WO 2000/42072 (Presta) describes antibody variants with increased or decreased FcR binding. See also, for example, Shields et al. J Biol. Chem. 9(2):6591-6604 (2001).

[0127] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform an ADCC effector function. Examples of human leukocytes that carry out ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; with PBMC and NK cells being preferred. The effector cells may be isolated from a natural source such as blood.

[0128] "Complement dependent cytotoxicity" or "CDC, CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) associated with the antigen it recognizes. To evaluate complement activation, a CSC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Variants of polypeptides with altered Fc region amino acid sequences (variant Fc region polypeptides) and increased or decreased ability to bind C1q are described, for example, in US Pat. No. 6,194,551 B1 and WO 1999/51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0129] The term “antibody containing an Fc region” refers to an antibody that includes an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during purification of the antibody or by recombinant construction of the antibody encoded by the nucleic acid. Thus, a composition containing an Fc-region-containing antibody according to the present invention may include an antibody containing K447, an antibody from which K447 has been completely removed, or a mixture of antibodies containing and not containing a K447 residue.

[0130] The terms "B cell surface marker" or "B cell surface antigen" as used herein refers to an antigen expressed on the surface of a B cell that can be targeted by an antagonist that binds to it, including but not limited to them, antibodies to the B-cell surface antigen or a soluble form of the B-cell surface antigen, capable of antagonizing the binding of the ligand to the native B-cell antigen. Examples of B cell surface markers include leukocyte surface markers CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 (for a description see The Leukocyte Antigen Facts Book, 2nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B cell surface markers include RP105, FcRH2, B cells CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. A B cell surface marker of particular interest is expressed preferentially on B cells over other non-B cell mammalian tissues, and it can be expressed on both progenitor B cells and mature B cells. .

[0131] The terms "malignancy" and "malignant"

refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of malignancies include, but are not limited to, hematopoietic malignancies or blood-related malignancies such as lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as splenic and lymph node malignancies, as well as carcinoma, blastoma, and sarcoma . More specific examples of malignancies include B-cell-associated malignancies, including, for example, well-differentiated, intermediate, and low-grade lymphomas (including B-cell lymphomas such as, for example, mucosal-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, and T-cell lymphomas) and leukemias (including secondary leukemia, chronic lymphocytic leukemia (CLL) such as B-cell leukemia (CD5+ B lymphocytes), myeloid leukemia such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia such as acute lymphoblastic leukemia (ALL) and myelodysplasia), and other hematologic and /or associated with B cells or T cells malignant neoplasms. Also included are hematopoietic accessory cell malignancies, including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells. Also included are malignant disorders of B cell proliferation selected from the following: lymphomas, non-Hodgkin's lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), DLBCL, relapsed/refractory DLBCL, FL, and mantle cell lymphoma. Sources of B-cell malignancies include the following: Marginal zone B-cell lymphoma originates from memory B cells in the marginal zone, follicular lymphoma and diffuse large B-cell lymphoma originate from centrocytes in the light zone of the germinal centers, chronic lymphocytic leukemia and small lymphocytic leukemia originate from B1 cells (CD5+), mantle cell lymphoma is derived from naive B cells in the mantle zone, and Burkitt's lymphoma is derived from centroblasts in the dark zone of germinal centers. Tissues that include hematopoietic cells, referred to herein as "hematopoietic cell tissues", include the thymus and bone marrow, and peripheral lymphoid tissues such as the spleen, lymph nodes, mucosal-associated lymphoid tissues such as gut-associated lymphoid tissues, tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucous membranes, such as the lining of the bronchi. Other specific examples of such malignancies include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, peritoneal malignancy, hepatic cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer , liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, cancer of the female vulva, cancer thyroid, liver carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0132] "B-cell malignancy" or "disorder of B-cell proliferation" as used herein includes non-Hodgkin's lymphoma (NHL), including low-grade/follicular NHL, small lymphocytic (ML) NHL, intermediate-differentiated/follicular NHL, intermediate -differentiated diffuse NHL, well-differentiated immunoblastic NHL, well-differentiated lymphoblastic NHL, well-differentiated small cell uncleaved NHL, massively affected NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Waldenström's macroglobulinemia; non-Hodgkin's lymphoma (NHL), DLBCL, relapsed/refractory DLBCL, lymphocyte-predominant Hodgkin's lymphoma (NLHL), small lymphocytic lymphoma (MLL), chronic lymphocytic leukemia (CLL), indolent NHL, including recurrent sluggish NHL and rituximab-refractory indolent NHL; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloid leukemia; lymphoma of cells of the mantle zone; and other hematological malignancies. Such malignancies can be treated with antibodies directed against B-cell surface markers such as CD79b. It is contemplated herein that such diseases can be treated by administering an antibody directed to a B-cell surface marker such as CD79b, and this includes administering an unconjugated ("naked") antibody or an antibody conjugated to a cytotoxic agent as described herein. . It is also contemplated herein that such diseases may be treated with a combination therapy comprising an anti-CD79b antibody or an anti-CD79b antibody drug conjugate of the invention in combination with another antibody or antibody drug conjugate, another cytotoxic agent, radiation therapy, or another method. treatment, carried out simultaneously or sequentially. In an exemplary method of treatment according to the invention, an anti-CD79b antibody according to the invention is administered in combination with an anti-CD20 antibody, an immunoglobulin, or a 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 invention and the anti-CD20 antibody is Rituxan® (rituximab).

[0133] The term "non-Hodgkin's lymphoma" or "NHL", as used herein, refers to a malignant neoplasm of the lymphatic system other than Hodgkin's lymphoma. Hodgkin's lymphomas can be distinguished from non-Hodgkin's lymphomas mainly by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and the absence of these cells in non-Hodgkin's lymphomas. Examples of non-Hodgkin's lymphomas that are encompassed by the term used herein include any lymphomas that a person skilled in the art (e.g., an oncologist or pathologist) can define as non-Hodgkin's lymphoma according to classification systems known in the art, such as the Revised European - American classification of lymphomas (REAL) as described in Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). See in particular FIG. 11.57, 11.58 and 11.59. More specific examples include, but are not limited to, relapsed or refractory NHL, borderline low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, lymphoblastic leukemia and/or progenitor B-cell 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 lymphoplasmic lymphoma, B-cell marginal zone lymphoma, marginal zone lymphoma of the spleen, extranodal marginal zone MALT lymphoma, nodular marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low-grade/follicular lymphoma, intermediate-differentiated/follicular NHL, mantle cell lymphoma, central follicle (follicular) lymphoma, intermediate-differentiated diffuse NHL, diffuse large B-cell lymphoma, relapsed/refractory diffuse large B-cell lymphoma, aggressive NHL (including borderline NHL with aggressive course and recurrent NHL with aggressive course), NHL relapsed after autologous stem cell transplantation or resistant to it, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, well-differentiated immunoblastic NHL, well-differentiated lymphoblastic NHL, well-differentiated small cell non-cleaved cell NHL, massive NHL, Burkitt's lymphoma, lymphoblastic leukemia and/or large granular progenitor lymphocytes (peripheral) lymphoma, mycosis fungoides and/or Cesari's syndrome, cutaneous lymphomas (cutaneous), anaplastic large cell lymphoma, angiocentric lymphoma.

[0134] A "disorder" is any condition that may benefit from treatment with a compound/molecule or method according to the invention. It includes chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated in accordance with this invention include malignant conditions such as malignant and benign tumors; non-leukemia and lymphoid malignancies; neuronal, glial, astroglial, hypothalamic and other glandular disorders, macrophage, epithelial, stromal and blastocele disorders; and inflammatory, immunological and other angiogenesis-related disorders. In addition, disorders include conditions such as B-cell proliferative disorders and/or B-cell tumors, e.g., lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent 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.

[0135] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with abnormal, to some extent, cell proliferation. In one embodiment, the cell proliferative disorder is a malignancy. In some embodiments of the present invention, the cancer is a disorder of B cell proliferation.

[0136] The term "tumor", as used herein, refers to any neoplastic cell growth and proliferation, both malignant and benign, and any premalignant and malignant cells and tissues.

[0137] The terms "treatment" or "treatment" or "mitigation" refers to both therapeutic and prophylactic or preventive measures, where the goal is to prevent or slow down (reduce) the intended pathological condition or disorder. Those in need of treatment include those who already have the disorder, as well as those who are predisposed to the disorder, or those whose disorder is to be prevented. A subject or mammal is successfully "treated" for a cancer expressing a CD79b polypeptide if, after obtaining a therapeutic amount of an anti-CD79b antibody using the methods of the present invention, the patient exhibits a marked and/or measurable reduction or absence of one or more of the following: a decrease in the number of malignant cells or the absence of malignant cells; reduction in tumor size; inhibition (ie, slowing down to some extent and preferably stopping) the infiltration of malignant cells into peripheral organs, including the spread of malignant tumors into soft tissue and bone; inhibition (ie, slowing down to some extent and preferably stopping) tumor metastasis; inhibition, to some extent, tumor growth; and/or mitigation, to some extent, of one or more symptoms associated with a particular cancer; reducing morbidity or mortality, and improving quality of life. Depending on whether the anti-CD79b antibody can prevent the growth and/or kill existing malignant cells, it can be cytostatic and/or cytotoxic. A decrease in these signs or symptoms may also be felt by the patient.

[0138] The above parameters for assessing successful treatment and improvement of the disease are easily determined by conventional methods known to the therapist. For cancer therapy, efficacy can be determined, for example, by evaluating time to disease progression (TTP) and/or determining response rate (RR). Metastasis can be determined by staging the disease and by scanning the bones and testing for calcium levels and other enzymes to determine spread to the bone. CT images can also be obtained to look for spread to the pelvis and lymph nodes in the area. Chest x-ray and measurement of liver enzyme levels by known methods are used to look for metastases in the lungs and liver, respectively. Other conventional methods for disease monitoring include transrectal ultrasound (TRUS) and transrectal needle biopsy (TRNB).

[0139] For bladder cancer, which is a more localized malignancy, methods for determining disease progression include urine cytology by cystoscopy, monitoring for blood in the urine, imaging of the urothelial tract by ultrasound or intravenous pyelography, computed tomography (CT), and magnetic resonance imaging. tomography (MRI). The presence of distant metastases can be assessed by abdominal CT, chest x-ray, or skeletal radionuclide imaging.

[0140] "Long-term" administration refers to the administration of the agent(s) continuously as opposed to a short-term regimen in order to maintain the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration is a treatment that is not administered without interruption, but is instead cyclic.

[0141] "Individual", "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.

[0142] The introduction "in combination with" one or more additional drugs includes simultaneous (joint) and sequential administration in any order.

[0143] The term "carriers", in the context of this document, includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cell or mammal exposed to them, in the dosages and concentrations used. Often the physiologically acceptable carrier is an aqueous pH buffer solution. 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) or PLURONICS®.

[0144] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to an anti-CD79b antibody immunoconjugate formulation that is shaped to provide the biological activity of the active ingredient and that does not contain additional components that are unacceptably toxic to the subject, to which the composition will be introduced. Such compositions are sterile. "Pharmaceutical" excipients (carriers, additives) are excipients that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient used.

[0145] A "sterile" composition is aseptic, free or substantially free of live microorganisms and their spores.

[0146] An "effective amount" of an anti-CD79b antibody-containing immunoconjugate disclosed herein is an amount sufficient to accomplish the specific stated purpose. An "effective amount" may be determined empirically and in conventional ways, depending on the intended purpose.

[0147] The term “therapeutically effective amount” refers to an amount of an anti-CD79b antibody-containing immunoconjugate effective to “treat” a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (ie, slow to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie, slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or alleviate to some extent one or more of the symptoms associated with the cancer. See the definition of "treatment" in this document. Depending on whether the drug can interfere with the growth and/or kill existing malignant cells, it can be cytostatic and/or cytotoxic. result. Typically, but not necessarily, as long as a prophylactic dose is used in individuals prior to or in the early stages of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0148] A "growth-inhibiting amount" of an anti-CD79b antibody-containing immunoconjugate is an amount capable of inhibiting the growth of a cell, especially a tumor cell, such as a cancer cell, either in vitro or in vivo. The "growth inhibitory amount" of an anti-CD79b antibody-containing immunoconjugate for the purpose of inhibiting neoplastic cell growth can be determined empirically and by conventional methods.

[0149] A "cytotoxic amount" of an anti-CD79b antibody-containing immunoconjugate is an amount capable of causing destruction of a cell, especially a tumor cell, such as a cancer cell, either in vitro or in vivo. The "cytotoxic amount" of an anti-CD79b antibody for the purpose of inhibiting neoplastic cell growth can be determined empirically and by conventional methods.

[0150] A "CD79b expressing cell" is a cell that expresses an endogenous or transfected CD79b polypeptide, either on the cell surface or in a secreted form. A "CD79b-expressing cancer" is a cancer that contains cells that have a CD79b polypeptide present on the cell surface, or that produce and secrete a CD79b polypeptide. A "CD79b-expressing cancer" does not necessarily produce sufficient levels of the CD79b polypeptide on its cell surface for an anti-CD79b antibody to bind to and exert a therapeutic effect on the cancer. In another embodiment, the "CD79b-expressing cancer" optionally produces and secretes sufficient levels of the CD79b polypeptide such that an anti-CD79b antagonist antibody can bind to it and have a therapeutic effect on the cancer. With respect to the latter, the antagonist may be an antisense oligonucleotide that reduces, inhibits or prevents the production and secretion of a secreted CD79b polypeptide by tumor cells. A cancer that "overexpresses" a CD79b polypeptide is a cancer that has, or produces and secretes, significantly higher levels of the CD79b polypeptide on its cell surface than a benign tissue cell of the same type. Such overexpression may be due to gene amplification or increased transcription or translation. Overexpression of a CD79b polypeptide can be determined in a detection or prognosis assay by evaluating elevated levels of CD79b protein present on the cell surface or secreted by the cell (e.g., by immunohistochemistry using anti-CD79b antibodies raised against an isolated CD79b polypeptide that can be obtained using recombinant DNA technology from the isolated nucleic acid encoding the CD79b polypeptide, FACS analysis, etc.). Alternatively or additionally, the levels of the CD79b polypeptide-encoding nucleic acid or mRNA in the cell can be measured, for example, by fluorescent in situ hybridization using a nucleic acid probe corresponding to the CD79b-encoding nucleic acid or its complementary sequence; (FISH; see W098/45479 published October 1998), Southern blot, Northern blot, or polymerase chain reaction (PCR) methods such as quantitative real-time PCR (RT-qPCR). It is also possible to investigate the overexpression of the CD79b polypeptide by measuring the antigen shedding from the cell in a biological fluid such as serum, for example, using antibody-based assays (also see, for example, US patent No. 4933294 issued June 12, 1990; W091/05264, published April 18, 1991, U.S. Patent 5,401,638, issued March 28, 1995, and Sias et al., J. Immunol Methods 132:73-80 (1990)). In addition to the above assays, various in vivo assays are available to those skilled in the art. For example, cells in the patient's body can be exposed to an antibody, which is optionally labeled with a detectable label, such as a radioactive isotope, and binding of the antibody to the patient's cells can be assessed, for example, by external scanning for radioactivity or by analyzing a biopsy taken from the patient for previously exposed to the antibody.

[0151] The term "cytotoxic agent", as used herein, refers to a substance that inhibits or interferes with cell function and/or causes cell destruction. The term is intended to include toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof capable of detrimental to cell growth or proliferation. In one embodiment, the cytotoxic agent is monomethylauristatin E (MMAE).

[0152] The term "lyophilized" composition, as used herein, refers to a liquid composition that has been freeze-dried during a freeze-drying process to form a "mass". In some embodiments, the lyophilized mass is stable under storage conditions (5° C. ± 3° C. and protected from light) without significant changes in structure, color, appearance, or moisture content. In a particular embodiment, the lyophilized mass is smooth without depressions under storage conditions. In some embodiments of this invention, the lyophilized composition is stable for about 60 months. In some embodiments of this invention, the lyophilized composition is stable for about 48 months.

[0153] A "stable" composition is one in which the immunoconjugate therein substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the composition substantially retains its physical and chemical stability as well as its biological activity upon storage. The shelf life is usually chosen depending on the expected shelf life of the drug. Various analytical methods for measuring protein stability are available in the art and are discussed in, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and/or quantitatively in a variety of different ways, including assessing the formation of aggregates (for example, using size exclusion chromatography, by measuring turbidity and/or visual control); by evaluating charge heterogeneity using cation exchange chromatography, image-controlled capillary isoelectric focusing (vcIEFv), or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; DNS-PAGE analysis to compare recovered and intact antibodies; peptide map analysis (eg tryptic or LYS-C); assessment of the biological activity or antigen-binding function of the antibody; and the like. Instability may include one or more of the following: aggregation, deamidation (for example, Asn deamidation), oxidation (for example, Met oxidation), isomerization (for example, Asp isomerization), clipping/hydrolysis/fragmentation (for example, fragmentation of the hinge region), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differences in glycosylation, etc.

[0154] An immunoconjugate "retains its physical stability" in a pharmaceutical composition if it shows no or very little evidence of aggregation, precipitation, and/or denaturation when visually examined for color and/or clarity, or when measured by UV light scattering or size exclusion chromatography.

[0155] An immunoconjugate "retains its chemical stability" in a pharmaceutical composition if the chemical stability at a given point in time is such that the immunoconjugate still retains its biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein portion of an immunoconjugate (eg, antibody). The chemical change may include size modification (eg, clipping), which can be assessed by, for example, size exclusion chromatography, DNS-PAGE and/or matrix-assisted laser desorption/ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical change include a change in charge (eg, resulting from deamidation), which can be assessed, for example, using ion exchange chromatography or ICEF. Additionally or alternatively, the chemical stability of the immunoconjugate can be assessed by detecting and quantifying chemically altered forms of the immunoconjugate drug moiety. Additionally or alternatively, the chemical stability of the immunoconjugate can be assessed by measuring the drug:antibody ratio (DAR), for example, using hydrophobic interaction chromatography (CHB) to determine the distribution of CALF in the composition containing the immunoconjugate.

[0156] An immunoconjugate antibody "retains its biological activity" in a pharmaceutical composition 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 is at least about 60% (within assay error) of the biological activity exhibited during the preparation of a pharmaceutical composition containing an immunoconjugate, as determined in an assay (eg, in an antigen binding assay).

[0157] A "deamidated" monoclonal antibody, as used herein, is one in which one or more of its asparagine residues have been derivatized with, for example, aspartic acid or isoaspartic acid.

[0158] An "oxidized" monoclonal antibody, as used herein, is an antibody in which one or more tryptophan residues and/or one or more of its methionines have been oxidized.

[0159] A "glycosylated" monoclonal antibody, as used herein, is an antibody in which one or more lysine residues are glycated.

[0160] An antibody that is "sensitive to deamidation" is an antibody containing one or more residues that have been found to be prone to deamidation.

[0161] An antibody that is "oxidation sensitive" is an antibody containing one or more residues that have been found to be susceptible to oxidation.

[0162] An antibody that is "subject to aggregation" is an antibody that has been found to form aggregates with other antibody molecule(s), especially when frozen and/or shaken.

[0163] An antibody that is "susceptible to fragmentation" is an antibody that has been found to cleave into two or more fragments, for example, in its hinge region.

[0164] By "reducing deamidation, oxidation, aggregation or fragmentation" is meant preventing or reducing the degree of deamidation, oxidation, aggregation or fragmentation compared to a monoclonal antibody included in another composition.

[0165] The antibody in the composition is preferably substantially pure and preferably substantially homogeneous (eg, free of contaminating proteins, etc.). "Substantially pure" antibody means a composition containing at least about 90% by weight of the antibody, based on the total weight of the proteins in the composition, preferably at least about 95% by weight. "Substantially homogeneous" antibody means a composition containing at least about 99% by weight of the antibody, based on the total weight of the proteins in the composition.

[0166] By "isotonic" is meant that the composition of interest has substantially the same osmotic pressure as human blood. Isotonic compositions typically have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured, for example, with a headspace osmometer or a freezing point osmometer.

[0167] The term "buffering agent", in the context of this document, refers to a buffered solution that resists changes in pH under the action of its acid-base conjugate components. Non-limiting examples of buffering agents herein include histidine, sodium phosphate, and sodium succinate. The buffering agent according to this document preferably has 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 buffering agent 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 buffering agent has a pH of 5.3. For example, sodium succinate is an example of a buffering agent that will control the pH in this range.

[0168] The term "surfactant", as used herein, refers to a surfactant, preferably a nonionic surfactant. Examples of surfactants herein include polysorbate (eg, polysorbate 20 and polysorbate 80); poloxamer (eg, poloxamer 188);

Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearylsarcosine; linoleyl-, myristyl- or cetylbetaine; lauroamidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmidopropyl or isostearamidopropyl betaine (eg lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyldimethylamine; disodium methyl cocoyl or methyl oleyl taurate; and the MONAQUATTM series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol and copolymers of ethylene and propylene glycol (eg Pluronics, PF68, etc.); and so on. In one embodiment, the surfactant herein is polysorbate 20. In another embodiment, the surfactant is polysorbate 80.

[0169] The term "sugar", as used herein, refers to soluble carbohydrates. Non-limiting examples of sugars include glucose, fructose, sucrose, trehalose, arginine, glycerin, proline, dextran, and sugar alcohols such as glycerol, mannitol, and sorbitol.

II. Pharmaceutical compositions

[0170] In one aspect, the invention provides a pharmaceutical composition comprising an immunoconjugate containing an anti-CD79b antibody and a surfactant, wherein the concentration of the surfactant is at least 0.06% w/v. (for example, 0.6 mg/ml), and while the immunoconjugate containing the antibody to CD79b has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, wherein the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5).

[0171] In some embodiments, the composition of this invention is lyophilized. In some embodiments, the composition disclosed herein is a reconstituted composition, that is, a composition that has been reconstituted from a lyophilized mass. In some embodiments, a 20 ml glass vial (eg, sealed vial) is provided that contains the pharmaceutical composition (eg, lyophilized composition or reconstituted composition) described herein.

A. Immunoconjugates

[0172] This invention also relates to immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADC") containing an antibody conjugated to a small molecular weight toxin such as monomethylauristatin (MMAE) (a synthetic analog of dolastatin). The present invention provides an immunoconjugate containing an anti-CD79b antibody of the formula Ab - (L - D)p, wherein Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, while the light chain contains (a) an HVR sequence -L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); L is a linker containing 6-maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (MC-val-cit-PAB); D is MMAE; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5); and while the immunoconjugate containing the antibody to CD79b has the structure:

An anti-CD79b immunoconjugate according to the invention may refer to 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 according to the invention. Polatuzumab vedotin has CAS registration number 1313206-42-6, IUPHAR/BPS number 8404, and KEGG D 10761. sale of an identical or bioequivalent drug in a country or territory selected from the group of countries consisting of the United States, European countries and Japan.

[0173] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is from about 5 mg/mL to about 60 mg/mL, from about 10 mg/mL to about 50 mg/mL, from about 10 mg /ml to about 40 mg/ml, from about 10 mg/ml to about 30 mg/ml, or from about 10 mg/ml to about 20 mg/ml. In some embodiment, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg /ml, about 11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml . 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.

[0174] In some implementations of any of the implementations presented herein, the "about" value or parameter covers a range of errors in any of the following values: ±10%, ±9%, ±8%, ±7%, ±6 %, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value or setting, including any range in between.

[0175] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is from about 10 mg/mL to about 20 mg/mL. In an exemplary embodiment, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is about 10 mg/mL. In another exemplary embodiment, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is about 20 mg/mL.

[0176] In some embodiments, the concentration of the anti-CD79b antibody-containing immunoconjugate in the pharmaceutical composition is 5 mg/mL to 60 mg/mL, 10 mg/mL to 50 mg/mL, 10 mg/mL to 40 mg /ml, 10 mg/ml to 30 mg/ml or 10 mg/ml to 20 mg/ml. In a specific embodiment, the concentration of the anti-CD79b immunoconjugate in the pharmaceutical composition is 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 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.

[0177] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is between 10 mg/mL and 20 mg/mL. In an exemplary embodiment, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is 10 mg/mL. In another illustrative embodiment, the immunoconjugate containing the anti-CD79b antibody is in the pharmaceutical composition at a concentration of 20 mg/mL.

[0178] As contemplated herein, an anti-CD79b antibody immunoconjugate is of Formula I as illustrated below, wherein the anti-CD79b antibody or fragment (Ab) is conjugated (i.e., covalently attached) to the MMAE drug moiety (D ) through the linker (L).

[0179] In Formula I, p is the average number of drug fragments per antibody, which can vary, for example, from about 1 to about 20 drug fragments per antibody, and in certain embodiments, from 1 to about 8 drug fragments per antibody. This invention relates to a composition comprising a mixture of antibody-drug compounds of compounds of Formula I, where the average drug load per antibody is from about 2 to about 5 or from about 3 to about 4, for example, 3.5.

1. Antibody to CD79b

[0180] This invention provides immunoconjugates containing an anti-CD79b antibody or functional fragments thereof.

[0181] In one aspect, the present invention provides an anti-CD79b antibody that preferably binds specifically to CD79b. Optionally, the antibody is a monoclonal antibody, an antibody fragment including a Fab, Fab', F(ab')2 and Fv fragment, 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 a polypeptide antibody to CD79b with its corresponding antigenic epitope. Antibodies of the present invention may optionally be produced in CHO cells or bacterial cells, and preferably induce the death of the cell to which they bind.

[0182] The present invention provides an immunoconjugate comprising an anti-CD79b antibody comprising a humanized anti-CD79b antibody, wherein the monovalent affinity of the antibody for CD79b (e.g., the affinity of the Fab antibody for CD79b) is substantially the same as the monovalent affinity of the mouse antibody (e.g. , the affinity of a mouse Fab antibody for CD79b) or a chimeric antibody (eg, the affinity of a chimeric Fab antibody for CD79b).

[0183] In one embodiment, the invention provides an anti-CD79b antibody immunoconjugate comprising a humanized anti-CD79b antibody, wherein the affinity of the antibody in its bivalent form for CD79b (e.g., the affinity of an IgG antibody for CD79b) is 0.3 nM or better. In another embodiment, the invention provides a humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form for CD79b (eg, the affinity of an IgG antibody for CD79b) is 0.5 nM. In a further embodiment, the invention provides a humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form for CD79b (eg, the affinity of an IgG antibody for CD79b) is 0.5 nM +/- 0.1. In another embodiment, the invention provides a humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form for CD79b (eg, the affinity of an IgG antibody for CD79b) is between 0.3 nM and 0.7 nM. In another embodiment, the invention provides a humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form for CD79b (eg, the affinity of an IgG antibody for CD79b) is between 0.4 nM and 0.6 nM. In another embodiment, the invention provides a humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form for CD79b (eg, the affinity of an IgG antibody for CD79b) is between 0.5 nM and 0.55 nM.

[0184] As is well known in the art, the binding affinity of a ligand to its receptor can be determined using any of a variety of assays and expressed in various quantitative terms. Accordingly, in one embodiment, the binding affinity is expressed as Kd values and reflects the binding affinity itself (eg, with minimized avidity effects). Typically, and preferably, binding affinity is measured in vitro, either under cell-free or cell-bound conditions. 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 the humanized antibody (e.g., Fab form) to the monovalent binding affinity value of the reference/comparative antibody (e.g., Fab form)( for example, a mouse antibody having hypervariable region donor sequences), wherein binding affinity values are determined under similar assay conditions. Thus, in one embodiment, the fold difference in binding affinity is defined as the ratio of the Kd values of a humanized antibody in Fab form and said reference/comparative Fab antibody. For example, in one embodiment, if an antibody of the present invention (A) has an affinity that is "3 times lower" than the reference antibody (M), then if the Kd value for A is 3x, the Kd value for M is 1×, and the ratio of Kd A to Kd M is 3:1. In contrast, in one embodiment, if the antibody of the present invention (C) has an affinity that is "3 times greater" than the reference antibody (R), then if the Kd value for C is 1×, the Kd value for R is 3×, and the ratio of Kd C to Kd R is 1:3. Any of a variety of assays known in the art can be used to obtain binding affinity scores, including assays described herein, including, for example, Biacore, radioimmunoassay (RIA) and ELISA.

[0185] The proposed antibody to CD79b contains a heavy chain and a light chain, while the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the HVR-H3 sequence TRRVPIRLDY (SEQ ID NO: 6).

[0186] In some embodiments of the invention, an anti-CD79b antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPG GGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYW GQGTLVTVSS (SEQ ID NO: 7)

and a light chain variable domain (VL) containing the amino acid sequence:

R(SEQ ID NO: 8).

[0187] In some embodiments, the heavy chain contains the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9)

and the light chain contains the amino acid sequence:

DIQLTQSPSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAA SNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 10).

2. Linker

[0188] This invention provides a linker that contains several components, including 6-maleimidocaproyl ("MC"), valine-citrulline ("val-cit" or "vc"), and p-aminobenzyloxycarbonyl ("PAB"). Such linker components are known in the art and are briefly described below.

[0189] The linker, in the context of this document, has Formula II:

[0190] where A is an extension element, and a is an integer from 0 to 1; W is an amino acid element, and w is an integer from 0 to 12; Y is a spacer element, and y is 0, 1, or 2. An "extension element" links the antibody to another linker component. The extension element provided herein, MC, is shown below (where the wavy line indicates the sites of covalent attachment to the antibody):

[0191] The amino acid element allows the cleavage of the linker by the protease, thereby facilitating the release of the drug from the immunoconjugate by intracellular proteases such as lysosomal enzymes. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21:778-784. The amino acid elements may contain naturally occurring amino acid residues as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. Amino acid elements can be designed and optimized for their selectivity for enzymatic cleavage by a particular enzyme, such as a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. The amino acid element provided herein is the dipeptide valine-citrulline (vc or val-cit).

[0192] The term "spacer" element, as used herein, is self-cleaving and binds an antibody to a drug moiety via an extension element and an amino acid element. The "self-cleaving" spacer element allows the release of the drug moiety without a separate hydrolysis step via an amide bond, and a carbamate, methyl carbamate, or carbonate is produced between the benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103. The spacer element provided herein is p-aminobenzyloxycarbonyl (PAB).

[0193] Linker components, including extension, spacer and amino acid elements, can be synthesized by methods known in the art, such as those described in US 2005-0238649A1.

[0194] In accordance with this invention, the linker is MC-val-cit-PAB, as shown below.

3. Monomethylauristatin (MMAE)

[0195] According to the invention, the immunoconjugate comprises an anti-CD79b antibody or fragment thereof conjugated to auristatin, a synthetic analog of dolastatin (US Pat. Nos. 5,635,483; 5,780,588).

Dolastatins and auristatins have been shown to disrupt microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anti-cancer activity (U.S. Patent No. 5663149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug moiety can be linked to the antibody via the N(amino)-terminus or C(carboxyl)-terminus of the peptide drug moiety (WO 02/088172). In accordance with the present invention, uristatin is a synthetic uncharged monomethylauristatin pentapeptide (MMAE) linked to the N-terminus as shown below.

[0196] An anti-CD79b antibody immunoconjugate of Formula I comprising an anti-CD79b antibody or fragment thereof, a linker and various linker components, and MMAE is illustrated below with the following abbreviations (where "Ab" is an anti-CD79b antibody or fragment thereof; p is 1 to about 8 (eg, about 2 to about 5, eg, about 3.5), "Val-Cit" or "vc" is a valine-citrulline dipeptide, and "S" is a sulfur atom.

[0197] It should be noted that in certain descriptions of the structure of the sulfur-bound immunoconjugate herein, the antibody is represented as "Ab-S", only to indicate the presence of a connection through sulfur, and not to indicate that a particular sulfur atom carries multiple linker-drug groups. The left parenthesis in the following structures may also be placed to the left of the sulfur atom, between Ab and S, which is an equivalent description of the immunoconjugate of the invention as described herein.

4. Drug loading

[0198] Drug loading is represented by p, the average number of drug groups per antibody in a Formula I molecule. Drug loading can range from 1 to 20 drug groups (D) per antibody. Formula I CALPs comprise collections of antibodies conjugated to a number of drug moieties ranging from 1 to 20. The average number of drug moieties per antibody in CALP preparations after conjugation reactions can be characterized by conventional methods such as mass spectrometry, ELISA analysis, and HPLC. In addition, the quantitative distribution of CALP in p-values can also be determined. In some cases, separation, purification and characterization of homogeneous KALP, where p is a specific value for KALP with a different drug load, can be carried out by methods such as reverse phase HPLC or electrophoresis. Thus, pharmaceutical compositions of Formula I antibody-drug conjugates may be a heterogeneous mixture of such conjugates wherein the antibodies are linked to 1, 2, 3, 4 or more drug moieties.

[0199] For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, in the case where the binding is through a cysteine thiol group, as in the embodiments provided herein, the antibody may have only one or more cysteine thiol groups, or it may have only one or more sufficiently reactive thiol groups through which it can linker to be attached. In some embodiments, a higher drug load, eg, p > 5, may result in aggregation, insolubility, toxicity, or loss of cell-permeability of certain antibody-drug conjugates. In some embodiments, the drug load for KALP in accordance with this invention is in the range from 1 to about 8; about 2 to about 6; or about 3 to about 5, or about 3.5. Indeed, it has been shown that for certain KALPs, the optimal ratio of drug groups to antibody may be less than 8, and it may be from about 2 to about 5. See US 2005-0238649 A1.

[0200] In some embodiments, fewer drug groups are conjugated during the conjugation reaction than the theoretical amount. The antibody may contain, for example, lysine residues that do not come into contact with the drug-linker intermediate or linker reagent, as discussed below. Typically, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to a drug moiety; indeed, most thiol cysteine residues in antibodies exist as disulfide bridges. In some embodiments, the antibody can be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partially or fully reducing conditions, to obtain reactive cysteine thiol groups. In some embodiments, the antibody is subjected to denaturing conditions to detect reactive nucleophilic groups such as lysine or cysteine.

[0201] The load (drug/antibody ratio) of CALP can be controlled in various ways, for example, by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) partial or limiting reducing conditions for modification of the thiol group of cysteine.

[0202] It should be understood that when more than one nucleophilic group comes into contact with a drug-linker intermediate or linker reagent, followed by reaction with the drug reagent, then the resulting product is a mixture of KALP compounds with one or more distributed drug groups associated with the antibody. The average number of drug moieties per antibody in a mixture can be calculated using a dual ELISA with antibodies that are antibody specific and drug specific. Individual CALP molecules can be identified in a mixture by mass spectrometry and separated by HPLC, eg, hydrophobic interaction chromatography (see, eg, McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al (2004) Clin Cancer Res 10:7063-7070 Hamblett, K. J., 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, S.C., 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). In some embodiments, homogeneous CALP with a single loading value can be isolated from a mixture of conjugates by electrophoresis or chromatography.

5. Exemplary methods for preparing immunoconjugates

[0203] CALP of formula I can be prepared in several ways using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) contacting an antibody nucleophilic group with a divalent linker reagent to form Ab-L via a covalent communication, followed by bringing into contact with the drug group D; and (2) contacting a nucleophilic group in the drug group with a divalent linker reagent, to form D-L, through a covalent bond, followed by contacting the nucleophilic group of the antibody. Exemplary methods for preparing CALP of Formula I using the latter method are described in US 2005-0238649 Al, which is incorporated herein by reference in its entirety.

[0204] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) hydroxyl or amino groups of sugars, where the antibody is glycosylated. The amino, thiol, and hydroxyl groups are nucleophilic and are capable of contacting to form covalent bonds with electrophilic groups on linker groups 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; (iii) aldehyde, ketone, carboxyl and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. The reactivity of the antibody for conjugation with linker reagents can be maintained by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the antibody is fully or partially reduced. Each cysteine bridge can thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by modifying lysine residues, for example by reacting lysine residues with 2-iminothiolane (Trot's reagent) to convert an amine to a thiol. Reactive thiol groups can be introduced into an antibody by inserting one, two, three, four or more cysteine residues (eg, by preparing antibody variants containing one or more non-native cysteine amino acid residues).

[0205] Antibody-drug conjugates of the present invention can also be prepared by contacting an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug. Suitable nucleophilic groups on the linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, the antibody is modified by introducing electrophilic groups that are capable of contacting nucleophilic substituents on the linker reagent or drug. In another embodiment, the sugars of glycosylated antibodies can be oxidized, for example, with periodate-based oxidizing reagents, to form aldehyde or ketone groups that can come into contact with the amino group of the linker reagents or drug groups. The resulting Schiff base imino groups can form a stable bond, or they can be reduced, for example, with borohydride reagents, to form stable bonds through an amine. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can result in carbonyl groups (aldehyde and ketone groups) in the antibody, which can react with the corresponding groups on the drug (Hermanson, Bioconjugate Techniques) . In another embodiment, antibodies containing N-terminal serine or threonine residues can come into contact with sodium metaperiodate, resulting in the formation of an aldehyde instead of the first amino acid (Geoghegan & Stroh (1992) Bioconjugate Chem. 3:138-146; US 5362852 ). Such an aldehyde can be reacted with a drug moiety or linker nucleophile.

[0206] Nucleophilic groups on a drug group include, but are not limited to: amino, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide groups capable of contacting to form covalent bonds with electrophilic groups on linker groups 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; (iii) aldehydes, ketones, carboxyl and maleimide groups.

[0207] Compounds of this invention include, but are not limited to, CALPs prepared with the following cross-linking 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, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A; see pages 467-498, 2003-2004 Applications Handbook and Catalog.

[0208] Immunoconjugates containing an antibody and a cytotoxic agent can also be prepared using various bifunctional protein attachment agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl )cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional imidoester derivatives (such as dimethyl adipimidate HC1), 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, the 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 a typical chelating agent for conjugating a radionuclide to an antibody. See W094/11026.

B. Surfactant

[0209] This invention provides a composition comprising an immunoconjugate containing an anti-CD79b antibody and a surfactant. In some embodiments, the surfactant is a polysorbate (eg, polysorbate 20 and polysorbate 80); poloxamer (eg, poloxamer 188); N-octyl-β-D glucopyranoside (OG);

Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearylsarcosine; linoleyl-, myristyl- or cetylbetaine; lauroamidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmidopropyl or isostearamidopropyl betaine (eg lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; the MONAQUATTM series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol, ethylene/propylene glycol copolymer such as Pluronics or PF68; or any combination of them.

[0210] In some embodiments, the surfactant is non-ionic. In some exemplary embodiments, the surfactant is selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188 (P188), N-octyl-β-D glucopyranoside (OG), and combinations thereof. In a particular embodiment, the surfactant is PS20. In another embodiment, the surfactant is PS80.

[0211] In certain embodiments, the concentration of a surfactant (eg, PS20) in the pharmaceutical composition is at least about 0.01% w/v. (ie, 0.1 mg/ml) and about 0.20% wt./about. (i.e., 2.0 mg/ml). In certain embodiments, the concentration of surfactant (eg, PS20) in the pharmaceutical composition is at least about 0.01% w/v. (ie, 0.1 mg/ml), at least about 0.02% wt./about. (ie, 0.2 mg/ml), at least about 0.03% wt./about. (ie, 0.3 mg/ml), at least about 0.04% wt./about. (ie, 0.4 mg/ml), at least about 0.05% wt./about. (ie, 0.5 mg/ml), at least about 0.06% wt./about. (ie, 0.6 mg/ml), at least about 0.07% wt./about. (ie, 0.7 mg/ml), at least about 0.08% wt./about. (ie, 0.8 mg/ml), at least about 0.09% wt./about. (ie, 0.9 mg/ml), at least about 0.10% wt./about. (ie, 1 mg/ml), at least about 0.11% wt./about. (ie, 1.1 mg/ml), at least about 0.12% wt./about. (ie, 1.2 mg/ml), at least about 0.13% wt./about. (ie, 1.3 mg/ml), at least about 0.14% wt./about. (ie, 1.4 mg/ml), at least about 0.15% wt./about. (ie, 1.5 mg/ml), at least about 0.16% wt./about. (ie, 1.6 mg/ml), at least about 0.17% wt./about. (ie, 1.7 mg/ml), at least about 0.18% wt./about. (ie, 1.8 mg/ml), at least about 0.19% wt./about. (ie, 1.9 mg/ml) or at least about 0.20% wt./about. (i.e., 2.0 mg/mL), including any range in between. In a specific embodiment, the concentration of surfactant (eg, PS20) in the pharmaceutical composition is at least about 0.06% w/v. (i.e., 0.6 mg/ml). In another specific implementation, the concentration of surfactant (for example, PS20) in the pharmaceutical composition is at least about 0.12% wt./about. (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 at least for about any of 24, 48 or 72 hours when stored at 2°C-8°C.

[0212] In certain embodiments, the concentration of surfactant (eg, PS20) in the pharmaceutical composition is at least about 0.01% wt./about. (ie, 0.1 mg/ml) and about 0.20% wt./about. (i.e., 2.0 mg/ml). In certain embodiments, the concentration of the surfactant (e.g., PS20) in the lyophilized pharmaceutical composition is at least 0.01% w/v, at least 0.02% w/v, at least 0 03% w/v, at least 0.04% w/v, at least 0.05% w/v, at least 0.06% w/v, at least at least 0.07% w/v, at least 0.08% w/v, at least 0.09% w/v, at least 0.10% w/v, at least 0.11% w/v, at least 0.12% w/v, at least 0.13% w/v, at least 0.14% w/v ., at least 0.15% wt./about., at least 0.16% wt./about., at least 0.17% wt./about., at least 0.18% of the mass. /about., at least 0.19% wt./about. or at least 0.20% wt./vol., including any range between these values. In a specific embodiment, the concentration of surfactant (eg PS20) in the lyophilized pharmaceutical composition is at least 0.06% w/v. In another specific implementation, the concentration of surfactant (for example, PS20) in the lyophilized pharmaceutical composition is at least 0.12% wt./about. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized mass. In some embodiments, the lyophilized pharmaceutical composition is contained in a glass vial.

[0213] In some embodiments, the concentration of the surfactant (eg, PS20) in the liquid pharmaceutical composition is at least about 0.1 to about 2.0 mg/mL. In some embodiments, the concentration of the surfactant (e.g., PS20) in the liquid pharmaceutical composition is at least any of 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 a particular embodiment, the concentration of the surfactant (eg, PS20) in the liquid pharmaceutical composition is at least about 0.6% mg/mL. In another specific embodiment, the concentration of surfactant (eg, PS20) in the liquid pharmaceutical composition is at least 1.2 mg/mL. In some embodiments, the liquid pharmaceutical composition is reconstituted (eg, reconstituted using CVD) from a lyophilized pharmaceutical composition (such as a lyophilized mass). 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 at least for about any of 24, 48 or 72 hours when stored at 2°C-8°C.

[0214] In an exemplary embodiment, the concentration of surfactant (eg, PS20) in the lyophilized pharmaceutical composition is 0.06% w/v. In another implementation, the concentration of surfactant (for example, PS20) in the lyophilized pharmaceutical composition is 0.12% wt./about. In some embodiments, the lyophilized pharmaceutical composition is a lyophilized mass. In some embodiments, the lyophilized pharmaceutical composition is contained in a glass vial.

[0215] In an exemplary embodiment, the concentration of surfactant (eg, PS20) in the liquid pharmaceutical composition is 0.6 mg/mL. In another exemplary embodiment, the concentration of surfactant (eg, PS20) in the liquid pharmaceutical composition is 1.2 mg/mL. In some embodiments, the liquid pharmaceutical composition is reconstituted (eg, reconstituted using CVD) from a lyophilized composition (such as a lyophilized mass). 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 at least for about 24, 48 or 72 hours when stored at 2-8°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 at least for about any of 24, 48 or 72 hours when stored at 2°C-8°C.

[0216] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is 10 mg/mL and the concentration of the surfactant in the pharmaceutical composition is at least 0.06% w/v. (i.e., 0.6 mg/ml). In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is 10 mg/mL and the concentration of the surfactant (eg, PS20) in the pharmaceutical composition is at least 0.12% w/v. (i.e., 0.12 mg/ml). In some embodiments, the pharmaceutical composition is a reconstituted composition (eg, reconstituted using CBID from a lyophilized composition). In some embodiments, the 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 at least for about 24, 48 or 72 hours when stored at 2-8°C.

[0217] In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody in the pharmaceutical composition is 10 mg/ml and the concentration of the surfactant (eg, PS20) in the pharmaceutical composition is 0.06% w/v. (i.e., 0.6 mg/ml). In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody in the pharmaceutical composition is 10 mg/mL and the concentration of the surfactant (eg, PS20) in the pharmaceutical composition is 0.12% w/v. (ie, 1.2 mg/ml). In some embodiments, the pharmaceutical composition is a reconstituted composition (eg, reconstituted using CBID from a lyophilized composition). In some embodiments, the pharmaceutical composition is contained in a glass vial.

[0218] In some embodiments, the concentration of the anti-CD79b antibody immunoconjugate in the pharmaceutical composition is 20 mg/mL and the concentration of the surfactant (eg, PS20) in the pharmaceutical composition is 0.06% w/v. (i.e., 0.6 mg/ml). In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody in the pharmaceutical composition is 20 mg/mL and the concentration of the surfactant (eg, PS20) in the pharmaceutical composition is 0.12% w/v. (i.e., 0.12 mg/ml). In some embodiments, the pharmaceutical composition is a reconstituted composition (eg, reconstituted using CBID from a lyophilized composition). In some embodiments, the pharmaceutical composition is contained in a glass vial.

[0219] In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody in the pharmaceutical composition is 20 mg/mL and the concentration of the surfactant (eg, PS20) in the pharmaceutical composition is 0.06% w/v. (i.e., 0.6 mg/ml). In some embodiments, the concentration of the immunoconjugate containing the anti-CD79b antibody in the pharmaceutical composition is 20 mg/mL and the concentration of the surfactant in the pharmaceutical composition is 0.12% w/v. (i.e., 1.2 mg/ml). In some embodiments, the liquid pharmaceutical composition is 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 at least for about 24, 48 or 72 hours when stored at 2-8°C.

[0220] In some embodiments, the pharmaceutical composition is a liquid composition suitable for intravenous administration. In some embodiments, a liquid composition suitable for administration contains from about 0.0432 to about 0.162 mg/ml of a surfactant (eg, polysorbate 20). In some embodiments, the liquid composition contains from about 0.72 mg/ml to about 2.7 mg/ml of polatuzumab vedotin. In some embodiments, a liquid composition suitable for intravenous administration contains about 0.72 mg/ml of polatuzumab vedotin and about 0.0432 mg/ml of polysorbate 20. In some embodiments, a liquid composition suitable for intravenous administration contains about 2 .7 mg/ml of Polatuzumab vedotin and about 0.162 mg/ml of Polysorbate 20.

C. Buffer agent

[0221] The present disclosure provides pharmaceutical compositions that include a buffering agent. Without being limited by theory, the use of a buffer maintains the pH of the pharmaceutical composition during the manufacture, storage and use of the composition. Non-limiting examples of buffering agents include histidine, succinate, sodium succinate, sodium bicarbonate, calcium chloride, magnesium sulfate, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl )amino)acetic acid (Bicine), 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), N-(2-hydroxy-1,1-bis(hydroxymethyl))ethyl)glycine (Tricine ), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazine- 1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsic acid, 2-morpholin-4-ylethanesulfonic acid (MES), or phosphate buffered saline (PBS). Other suitable buffering agents may be acetic acid and its salt, citric acid and its salt, boric acid and its salt, and phosphoric acid and its salt.

[0222] In exemplary embodiments of the present disclosure, the buffering agent is a histidine buffer or a succinate buffer. In a particular embodiment, the succinate buffer is sodium succinate buffer.

[0223] In some embodiments, the buffering agent is at a concentration of about 10 mM to about 200 mM. In a particular embodiment, the buffering agent 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 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.

[0224] In another specific embodiment, the buffering agent is at a concentration of 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM , 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mm, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, about 190 mM, 195 mM or 200 mM.

[0225] In an exemplary embodiment of the present disclosure, the concentration of sodium succinate buffer is from about 10 mM to about 200 mM. In one embodiment, the concentration of sodium succinate buffer is about 10 mM. In another embodiment, the concentration of sodium succinate buffer is 10 mM.

[0226] In some embodiments, the composition of the present disclosure has a pH of about 5.0 to about 6.0. In particular 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.

[0227] 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.

D. Sugar

[0228] In accordance with the present disclosure provides pharmaceutical compositions containing sugar. Without wishing to be bound by theory, the sugar functions to help cryoprotect the pharmaceutical composition, thereby preventing aggregation and maintaining the chemical and physical stability of the pharmaceutical composition. In some embodiments, the sugar is glucose, fructose, sucrose, trehalose, arginine, glycerol, proline, dextran, dextran 40, glycerol, mannitol, or sorbitol.

[0229] 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.

[0230] In some embodiments, the sugar concentration is from about 50 mM to about 300 mM. In a particular embodiment, the buffering agent concentration is about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM , about 280 mM, about 290 mM, or about 300 mM. In some embodiments, the sugar concentration is from about 100 mM to about 260 mM.

[0231] In some embodiments, the sugar concentration is between 50 mM and 300 mM. In a particular embodiment, the buffering agent concentration is 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 implementations; the sugar concentration is from 100 mM to 260 mM.

[0232] In an exemplary embodiment; the sucrose concentration is about 120 mM. In an exemplary embodiment, the concentration of sucrose is 120 mM.

[0233] In another aspect, the present disclosure provides a pharmaceutical composition obtained by lyophilization of a liquid composition containing 20 mg/ml of an immunoconjugate containing an anti-CD79b antibody in 10 mM sodium succinate buffer, 0.12% w/v. polysorbate 20 and 120 mm sucrose, and the liquid composition has a pH of 5.3, and the immunoconjugate containing the antibody to CD79b has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, wherein the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5).

[0234] In some embodiments, an anti-CD79b antibody of the present invention 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. B In some embodiments of the present invention, the heavy chain contains the amino acid sequence of SEQ ID NO: 9, and the light chain contains the amino acid sequence of SEQ ID NO: 10.

E. Stability

[0235] In some embodiments, the physical stability, chemical stability, or biological activity of a pharmaceutical composition in liquid state or lyophilized form is assessed or measured. To assess the stability and biological activity of the pharmaceutical composition of the present invention can be applied to any methods known in the art and described in the examples in this document. For example, the stability of an antibody in a pharmaceutical composition can be measured by, but not limited to, size exclusion chromatography (SEC or E-HPLC), image-guided capillary isoelectric focusing (IEF), peptide mapping, particulate analysis by low volume darkening light (HIAC) ) and variants of capillary electrophoresis (CE) methods such as sodium dodecyl sulfate-CE (CE-SDS) and glycan-CE. In some embodiments, a stable pharmaceutical composition (or formulation) is a composition in which the immunoconjugate substantially retains its physical stability and/or chemical stability and/or biological activity upon storage, such as storage for a specified period or length of time ( eg hours, days, months, years, etc.) under certain conditions (such as temperature, relative humidity, residual humidity, presence or absence of light, after a period of shaking, etc.). In some embodiments, an immunoconjugate present in a pharmaceutical composition is biologically stable (e.g., retains its biological activity) if the biological activity of the immunoconjugate (and/or an antibody fragment of an immunoconjugate, such as an anti-CD79b antibody) at a particular point in time is within the range approved by a national or regional regulatory agency for pharmaceutical products (e.g. US Federal Drug Administration (FDA), Australian Therapeutic Goods Administration (TGA), European Medicines Agency (EMA) of the European Union, etc.). For example, the biological activity of an immunoconjugate (such as an immunoconjugate containing an anti-CD79b antibody) or a pharmaceutical composition containing such an immunoconjugate can be measured by its ability to bind an antigen (such as the ability of an anti-CD79b antibody to bind CD79b, e.g., human CD79b). A number of assays known in the art can be used to measure antibody binding affinity, including but not limited to surface plasma resonance (SPR), radioimmunoassay (RIA), and ELISA. Additionally or alternatively, the biological activity of an immunoconjugate (such as an immunoconjugate containing an anti-CD79b antibody) is measured by its ability to inhibit cell growth and/or cell proliferation, for example, in vitro or in vivo, or its ability to induce cell death, including programmed cell death ( apoptosis), for example, in vitro or in vivo. Additional details regarding assays for measuring the biological activity of an anti-CD79b antibody or an immunoconjugate containing an anti-CD79b antibody are provided in US Pat. No. 8,088,387, the contents of which are incorporated herein by reference in their entirety.

[0236] In some embodiments, the pharmaceutical composition of the present disclosure is 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 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 protected from light.

[0237] In some embodiments, the pharmaceutical composition of the present disclosure is stable at 2°C to 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 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 protected from light.

[0238] In some embodiments, the pharmaceutical composition of the present disclosure is stable at 25°C to 40°C for at least about 30 minutes, at least about 60 minutes, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours , at least about 20 hours, at least about 22 hours, at least about 1 day, at least about 2 days, or at least about 3 days.

[0239] In one exemplary embodiment, the composition is stable for at least about 48 months at 5°C±3°C protected from light. In another exemplary implementation, the composition is stable for about 48 months at 5°C±3°C protected from light.

[0240] In some embodiments, the lyophilized form (eg, lyophilisate) of the pharmaceutical composition of the present invention may be tested for stability. In some embodiments, under storage conditions (5° C. ± 3° C. and protected from light), the lyophilized mass does not show any significant changes in structure, color, appearance, or moisture content. In one particular embodiment, the lyophilized mass has a moisture content of less than 5% under storage conditions. In another particular embodiment, the lyophilized mass is smooth without depressions under storage conditions. In some embodiments of the present invention, the lyophilized mass is stable for at least about 48 months under storage conditions.

[0241] In some embodiments, the stability of the pharmaceutical composition of the present invention is measured using size exclusion high performance liquid chromatography (E-HPLC). In an exemplary embodiment, the composition has a main peak (area %) of at least 95.0 as measured by e-HPLC.

[0242] In some embodiments, a lyophilized pharmaceutical composition (e.g., bulk) provided herein is stable for at least about 44 months (e.g., about 30, 35, 40, 45, 50, or 55 months) when stored at a temperature of from about 2°C to about 8°C in the range of residual moisture content from about 0.3% (wt./wt.) to about 3.2% (wt./wt.), including any range between these values . In some embodiments, a lyophilized pharmaceutical composition (e.g., bulk) provided herein is stable for at least about 7 months (e.g., about 2, 3, 4, 5, 6, 7, 8, 8 or 10 months) when stored at about 25°C in the range of residual moisture content from about 0.3% (wt./wt.) to about 3.2% (wt./wt.), including any range between these values.

[0243] In some embodiments, a lyophilized pharmaceutical composition (e.g., bulk) provided herein is stable at (a) a protein concentration of about 17 to about 23 mg/mL, (b) a succinate concentration of about 7 to about 23 mM, (c) a sucrose concentration of about 90 to about 150 mM, (d) a polysorbate 20 concentration 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, a lyophilized pharmaceutical composition (e.g., mass) comprising any one or more of (a) to (e) above is stable at about 2°C to about 8°C for at least about any of 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.

G. Compositions for intravenous administration.

[0244] The present disclosure provides pharmaceutical compositions and liquid compositions suitable for intravenous (IV) administration. Compositions intended for intravenous in vivo use must be sterile. This is easily achieved by filtration through sterile filter membranes before or after preparation of the pharmaceutical composition. In some embodiments, the pharmaceutical composition of the present disclosure is reconstituted with sterile water for injection (SWI). In some embodiments, the reconstituted composition is further diluted in isotonic buffer in an intravenous (IV) bag.

[0245] In one aspect, the present disclosure provides a pharmaceutical composition comprising an immunoconjugate comprising an anti-CD79b antibody, a surfactant, a succinate buffer, and a sugar, wherein the pharmaceutical composition, when reconstituted in water, forms a liquid pharmaceutical composition comprising an immunoconjugate comprising an anti-CD79b antibody. at a concentration of from about 10 mg/kg to about 20 mg/ml, a surfactant at a concentration of at least 0.06% wt./about. (i.e., 0.6 mg/mL), succinate buffer at a concentration of about 10 mM to about 200 mM, and sugar at a concentration of about 100 mM to about 260 mM, wherein the liquid pharmaceutical composition has a pH of 5.3, and an immunoconjugate containing an anti-CD79b antibody has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, wherein the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5).

[0246] In some embodiments, the pharmaceutical composition is reconstituted (1:1 weight:volume) in CBDI. In some embodiments, the pharmaceutical composition, when reconstituted in water, forms a liquid pharmaceutical composition containing 20 mg/ml of an immunoconjugate containing an anti-CD79b antibody in 10 mM sodium succinate buffer, 0.12% w/v. polysorbate 20 and 120 mm sucrose, while the liquid pharmaceutical composition has a pH of 5.3.

[0247] In some embodiments, the pharmaceutical composition is reconstituted in SIDS and then diluted with isotonic buffer in an IV bag, wherein the concentration of surfactant when diluted in the IV bag is at least 0.003% by weight. /about. In one embodiment, the concentration of the surfactant when diluted in the IV bag is at least 0.004% w/v. In an exemplary embodiment, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In a specific implementation, the concentration of sucrose is 120 mm. In some embodiments, the succinate buffer is sodium succinate buffer. In a specific exemplary embodiment, the concentration of sodium succinate buffer is 10 mM.

[0248] In some embodiments, the stability of a pharmaceutical composition of the present disclosure when reconstituted in water is measured by aggregation, pH, turbidity, visible and/or invisible particles, or free (eg, unconjugated) drug. In some embodiments, UV spectrophotometry, E-HPLC, hydrophobic interaction chromatography (HIC), hcIEF, or HIAC, or a combination thereof, can be used to assess stability. In some embodiments, the pharmaceutical composition, when reconstituted in water, is stable for up to about 1 day, up to about 2 days, or up to about 3 days at 30°C. In some embodiments, the pharmaceutical composition, when reconstituted in water, is stable for 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. C ± 3°C.

[0249] In some embodiments, the stability of the composition reconstituted in water is measured using size exclusion high performance liquid chromatography (E-HPLC). In an exemplary embodiment, the composition has a main peak (area %) of at least 95.0 as measured by e-HPLC.

[0250] In some specific embodiments, the composition has an amount of HMC (%) less than 4.0, as measured by e-HPLC. In some specific embodiments, the composition has an amount of HMS (%) less than 1.0 as measured by E-HPLC. In some embodiments, the composition has a main peak (area %) of at least 95.0 and a BMC of less than 4.0 as measured by e-HPLC. In some embodiments, the composition has a main peak (area %) of at least 95.0, a HMW (%) of less than 4.0, and an HMW (%) of less than 1.0, as measured by e-HPLC.

[0251] In some embodiments, the stability of the composition reconstituted in water is measured using image-controlled capillary isoelectric focusing (ICEF). In one embodiment, the composition has a main peak (area %) of at least 73.0, an acid region (area %) of no more than 25.5, and a main region (area %) of no more than 10.0, as measured by VKIEF. In an exemplary embodiment, the composition has a main peak (area %) of at least 58.0, an acid region (area %) of no more than 32.0, and a main region (area %) of no more than 12.0, as measured by VKIEF.

[0252] In some embodiments, a reconstituted pharmaceutical composition that comprises 20 mg/mL Polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, 1.2 mg/mL polysorbate 20 at pH 5.3 is physicochemically stable after 72 hours storage at a temperature of from about 2°C to about 8°C. In some embodiments, storage of the reconstituted pharmaceutical composition for 72 hours at a temperature in the range of about 2°C to about 8°C does not reduce the biological activity of polatuzumab vedotin. In some embodiments, a reconstituted pharmaceutical composition that comprises 20 mg/mL Polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, 1.2 mg/mL polysorbate 20 at pH 5.3 is physicochemically stable after 24 hours of storage at 30°C with ambient lighting. In some embodiments, storage of the reconstituted pharmaceutical composition for 24 hours at 30° C. with exposure to ambient light does not reduce the biological activity of polatuzumab vedotin.

[0253] In one aspect, the present disclosure provides a liquid

a composition containing an immunoconjugate containing an anti-CD79b antibody, a surfactant, a succinate buffer and a sugar, wherein the immunoconjugate containing the anti-CD79b antibody is at a concentration of about 10 mg/ml to about 20 mg/ml, the surfactant is at a concentration at least 0.06% wt./about. (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, with the liquid composition having a pH of 5.3, and at This immunoconjugate containing the antibody to CD79b has the formula:

Where

Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, wherein the light chain contains (a) the sequence of HVR-L1 KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); and (c) the HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); wherein the heavy chain contains (a) the sequence HVR-H1 GYTFSSYWIE (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); and (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p is about 1 to about 8 (eg, about 2 to about 5, eg, about 3.5).

[0254] In some embodiments, the surfactant is polysorbate 20. In some embodiments, the sugar is sucrose. In an exemplary embodiment, the concentration of sucrose is 120 mM. In some embodiments, the succinate buffer is sodium succinate buffer. In a specific exemplary embodiment, the concentration of sodium succinate buffer is 10 mM.

[0255] In some embodiments, the liquid composition of the present disclosure is dissolved in a buffer. The buffer can be, without limitation, saline (0.9% w/v sodium chloride), half saline (0.45% w/v sodium chloride), 5% w/v. dextrose, Ringer's lactate solution, or a combination thereof.

[0256] In some embodiments, the liquid composition of the present disclosure is dissolved in an isotonic buffer. In some embodiments, the isotonic buffer may be freshly prepared or contained in a pre-filled IV bag. In some embodiments, the isotonic buffer is saline (0.9% sodium chloride). In an exemplary embodiment, the liquid composition of the present disclosure, dissolved in isotonic buffer, is in an IV bag.

[0257] In some embodiments, the liquid composition of the present disclosure, dissolved in isotonic buffer in an IV bag, has a pH of about 4.8 to about 5.8. In particular embodiments, 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 isotonic buffer in an IV bag, has a pH of about 5.3.

[0258] In some embodiments, the stability of the liquid composition in the IV bag of the present disclosure is measured by aggregation, pH, turbidity, visible and/or invisible particles, or free (e.g., unconjugated) drug. In some embodiments, UV spectrophotometry, E-HPLC, hydrophobic interaction chromatography (HIC), hcIEF, or HIAC, or a combination thereof, may be used to evaluate the stability of a liquid formulation in an IV pouch.

[0259] In some embodiments, the liquid composition in the IV pouch of the present disclosure is stable under static conditions. In some embodiments, the liquid composition in the IV sachet of the present disclosure is stable under shaking conditions. In some embodiments, the liquid composition of the present disclosure, dissolved in isotonic buffer in an IV bag, is stable for 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 isotonic buffer in an IV bag, is stable for 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 isotonic buffer in an IV bag, is stable for 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 some embodiments, the isotonic buffer is saline.

[0260] In some embodiments, the reconstituted solution or diluted infusion solution described herein (eg, diluted infusion solution containing an immunoconjugate (eg, Polatuzumab vedotin)) is sterile.

III. Methods of treatment

[0261] It is contemplated that the pharmaceutical composition or liquid composition of the present disclosure may be used to treat various proliferative disorders, such as those characterized by tumor antigen overexpression. An example of a proliferative disorder is a malignant neoplasm. Examples of malignancies include, but are not limited to, hematopoietic malignancies or blood-related malignancies such as lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as splenic and lymph node malignancies, as well as carcinoma, blastoma, and sarcoma . More specific examples of malignancies include disorders of B cell proliferation, including, for example, high-grade, intermediate, and low-grade lymphomas (including B-cell lymphomas such as, for example, mucosal-associated B-cell lymphoma and non-Hodgkin's lymphoma ( NHL), mantle cell lymphoma, Burkitt's lymphoma, small cell lymphocytic lymphoma, marginal zone lymphoma, diffuse large B cell lymphoma, follicular lymphoma, Hodgkin's lymphoma and T-cell lymphomas) and leukemias (including secondary leukemia, chronic leukemia leukemia (CLL), such as B-cell leukemia (CD5+ B-lymphocytes), myeloid leukemia such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia such as acute lymphoblastic leukemia (ALL) and myelodysplasia) and other hematologic and/or B-associated cells or T cells malignant neoplasms. Also included are hematopoietic accessory cell malignancies, including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells. In some embodiments, the proliferative disorder of the present disclosure is a relapsed cancer or a refractory cancer.

[0262] In some embodiments, the pharmaceutical composition or liquid composition of the present disclosure may be used to treat recurrent leukemia or recurrent lymphoma. In other specific embodiments, the pharmaceutical composition or liquid composition of the present disclosure may be used to treat refractory leukemia or refractory lymphoma.

[0263] In one aspect, the present disclosure provides a method for treating a proliferative disorder in a patient in need thereof, comprising administering to the patient a pharmaceutical composition or liquid composition described herein.

[0264] In some embodiments, the cancer is a disorder of B cell proliferation. In specific embodiments, the B cell proliferation 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), recurrent aggressive NHL, relapsed indolent NHL, relapsed NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0265] In one exemplary embodiment of the present disclosure, the B cell proliferation disorder is non-Hodgkin's lymphoma (NHL). In one exemplary embodiment, the B cell proliferation disorder is diffuse large B cell lymphoma (DLBCL). In one specific embodiment, the disorder of B cell proliferation is recurrent diffuse B cell lymphoma (DLBCL). In another specific embodiment, the B cell proliferation disorder is refractory diffuse B cell lymphoma (DLBCL).

[0266] In one exemplary embodiment, the B cell proliferation disorder is relapsing NHL or refractory NHL. In yet another exemplary embodiment, the B cell proliferation disorder is follicular lymphoma.

[0267] The cancer may contain cells expressing CD79b such that the anti-CD79b antibody-containing immunoconjugates of the present disclosure are able to bind to the cancer cells. Various diagnostic/prognostic assays are available to determine CD79b expression in cancer. In one embodiment, CD79b overexpression can be analyzed by IHC. Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC analysis and meet the CD79b protein staining intensity criteria in terms of the degree of staining and what proportion of the examined tumor cells are present in the sample.

[0268] For the prevention or treatment of a disorder, an appropriate dose of CALP will depend on the type of disease being treated, as defined above, the severity and course of the disease, whether the molecule is being administered prophylactically or therapeutically, early therapies, the patient's medical history, and response on the antibody, as well as at the discretion of the attending physician. The compound is conveniently administered to a patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage of a compound for administration to a patient may be from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg - 20 mg/kg), for example, for one or more separate injections or as a continuous infusion. One typical daily dosage may range from about 1 μg/kg to 100 mg/kg or more, depending on the aforementioned factors. An exemplary dose of CALP administered to a patient is in the range of about 0.1 to about 10 mg/kg of the patient's body weight. With repeated administration for several days or longer, depending on the condition, treatment is continued until the desired suppression of the symptoms of the disease.

[0269] To treat or prevent a proliferation disorder, the pharmaceutical composition or liquid composition of the present disclosure is administered by intravenous infusion. The dose administered by infusion is in the range of about 1 μg/m2 to about 10,000 μg/m2 per dose, typically one dose per week, for a total of one, two, three or four doses. Alternatively, the dose is in the range of about 1 µg/m2 to about 1000 µg/m2, about 1 µg/m2 to about 800 µg/m2, about 1 µg/m2 to about 600 µg/m2, about 1 µg/m2 m2 to about 400 µg/m2, from about 10 µg/m2 to about 500 µg/m2, from about 10 µg/m2 to about 300 µg/m2, from about 10 µg/m2 to about 200 µg/m2, and from about 1 µg/m2 to about 200 µg/m2. The dose can be administered once a day, once a week, several times a week, but less than once a day, several times a month, but less than once a day, several times a month, but less than once a week, one once a month, or periodically to relieve or reduce symptoms of the disease. Administration may be continued at any of the indicated intervals until remission of the tumor or symptoms of the lymphoma of the leukemia to be treated. Administration may be continued after remission or relief of symptoms is achieved if such remission or relief is prolonged by such prolonged administration.

[0270] Various detection assays are available to determine CD79b expression in cancer. In one embodiment, overexpression of a CD79b polypeptide can be analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC analysis and meet the following CD79b staining intensity criteria:

Score 0 - No staining or membrane staining is seen in less than 10% of the tumor cells.

Score 1+ - weak/hardly noticeable membrane staining is found in more than 10% of tumor cells. Cells are stained only in part of their membrane.

Score 2+ - complete staining of the membrane from weak to moderate is observed in more than 10% of tumor cells.

Score 3+ - complete moderate to strong membrane staining is observed in more than 10% of tumor cells.

[0271] Tumors with a score of 0 or 1+ for CD79b polypeptide expression can be characterized as not overexpressing CD79b, while tumors with scores of 2+ or 3+ can be characterized as overexpressing CD79b.

[0272] Alternatively or additionally, FISH assays such as INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois) can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of overexpression CD79b in the tumor.

[0273] CD79B overexpression or amplifications can be assessed using an in vivo detection assay, e.g., by introducing a compound (such as an antibody) that binds the detection compound and is labeled with a detectable label (e.g., a radioactive isotope or fluorescent label) and an external scan patient to determine the location of the label.

Combination Therapies

[0274] Currently, depending on the stage of the cancer, treatment of cancer includes one or a combination of the following treatments: surgery to remove cancer tissue, radiation therapy, and chemotherapy. Therapy with anti-CD79b immunoconjugates may be particularly desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy, and in metastatic disease where radiotherapy is of limited utility. The tumor-targeting anti-CD79b immunoconjugates of the present disclosure are useful in alleviating CD79b-expressing cancer at initial disease diagnosis or during relapse. For therapeutic use, an immunoconjugate containing an anti-CD79b antibody can be used alone or in combination therapy, for example with hormones, antiangiogenic agents, or radiolabeled compounds, or with surgery, cryotherapy, and/or radiotherapy. Administration of the pharmaceutical composition or liquid composition of the present disclosure may be in combination with other forms of conventional therapy, or sequentially, before or after conventional therapy. Chemotherapeutic drugs such as TAXOTERE® (docetaxel), TAXOL® (paclitaxel), estramustine and mitoxantrone are used to treat cancer, particularly in high-risk patients. In the present method of the present disclosure, an anti-CD79b antibody may be administered in combination with treatment with one or more prior chemotherapeutic agents to treat or alleviate cancer in a patient with cancer. In particular, combination therapy with paclitaxel and modified derivatives is contemplated (see, for example, EP 0600517). The pharmaceutical composition or liquid composition of the present disclosure may be administered with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, the pharmaceutical composition or liquid composition of the present disclosure is administered in combination with chemotherapy to enhance the activity and efficacy of a chemotherapeutic agent, such as paclitaxel. The Physicians' Desk Reference (PDR) describes dosages of these agents that are used to treat various types of cancer. The administration schedule and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular type of cancer being treated, the extent of the disease, and other factors familiar to the physician in the art, and may be determined by the physician.

[0275] In another embodiment, the pharmaceutical composition or liquid composition of the present disclosure may be administered in combination with one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents or other cytotoxic agent(s) or other therapeutic agent (c), which also inhibits tumor growth. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Formulations and regimens for administering such chemotherapeutic agents may be used according to the manufacturer's instructions or as determined empirically by one of skill in the art. In addition, formulations and administration schedules for such chemotherapeutic agents are described in "Chemotherapy Service," Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The pharmaceutical composition or liquid composition of the present disclosure may be combined with an anti-hormonal compound; for example, an antiestrogen compound such as tamoxifen; an antiprogesterone such as onapristone (see EP 616812); or an antiandrogen such as flutamide at dosages known for such compounds. If the cancer to be treated is androgen-independent cancer, the patient may have previously been subjected to antiandrogen therapy, and after the cancer becomes androgen-independent, the pharmaceutical composition or liquid composition of the present invention may be administered to the patient.

[0276] Sometimes it may also be useful to simultaneously administer to the patient a cardioprotector (to prevent or reduce therapy-related myocardial dysfunction) or one or more cytokines. In addition to the above therapeutic regimens, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy (for example, external beam radiation or therapy with a radioactively labeled agent such as an antibody) before, simultaneously with or after administration of the pharmaceutical composition or liquid composition of the present invention. Suitable dosages for any of the above co-administered agents are those currently in use and can be reduced by the combined action (synergy) of the agent and the anti-CD79b antibody.

[0277] In some embodiments, a pharmaceutical composition or liquid composition of the present disclosure may be administered to a patient in combination with an effective amount of another therapeutic agent. In one embodiment, the therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, a cytotoxic agent, an antiangiogenic agent, an immunosuppressant, a prodrug, a cytokine, a cytokine antagonist, a cytotoxic radiotherapy, a corticosteroid, a cancer vaccine, and a growth inhibitory agent. In another embodiment, the therapeutic agent is selected from one or more of the following, 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, prednisolone, and docetaxel.

[0278] In some embodiments, the therapeutic agent is an anti-CD20 antibody.

[0279] In some embodiments, a pharmaceutical composition or liquid composition of the present disclosure may be administered in combination with an effective amount of an anti-CD20 antibody to treat a disorder of B cell proliferation. In one embodiment, the B cell proliferation disorder is diffuse large B cell lymphoma (DLBCL). In another embodiment, the proliferation disorder is follicular lymphoma (FL). In some embodiments, the anti-CD20 antibody is rituximab.

[0280] In some embodiments, the therapeutic agent is a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent comprises one or more of the following: cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone. In another embodiment, the chemotherapeutic agent includes cyclophosphamide, doxorubicin, and prednisone.

[0281] In some embodiments, the pharmaceutical composition 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. In one embodiment, the chemotherapeutic agent includes cyclophosphamide, doxorubicin, and prednisone.

[0282] In some embodiments, a pharmaceutical composition 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 disorder of B cell proliferation. In one embodiment, the B cell proliferation disorder is non-Hodgkin's lymphoma (NHL). In another embodiment, the B cell proliferation disorder is diffuse large B cell lymphoma (DLBCL). In a particular embodiment, DLBCL is relapsed/refractory DLBCL. In another embodiment, the NHL is relapsing NHL or refractory NHL. In another specific embodiment, the proliferation disorder is follicular lymphoma (FL). In another embodiment, FL is relapsing/refractory FL. In some embodiments, the anti-CD20 antibody is rituximab.

[0283] In some embodiments, the pharmaceutical composition or liquid composition of the present disclosure may be combined with an anti-CD20 antibody (either naked antibody or KALP). 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 composition or liquid composition of the present disclosure may be combined with an anti-VEGF antibody (eg, Avastin®).

[0284] In some embodiments, the pharmaceutical composition or liquid composition of the present disclosure may be combined with an alkylating agent. In some embodiments, a pharmaceutical composition or liquid composition of the present disclosure may be combined with an alkylating agent and an anti-CD20 antibody (either naked antibody or KALP). In some embodiments, the anti-CD20 antibody is rituximab (Rituxan®) or a biosimilar thereof. In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl ]butanoic acid and its salts. In some embodiments, the alkylating agent is bendamustine.

[0285] Combination therapy can be used according to a simultaneous or sequential administration schedule. When administered sequentially, the combination may be administered over two or more administrations. Combined administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in any order, and it is preferable that there is a period of time when both (or all) active agents simultaneously exhibit their biological activity.

[0286] Suitable dosages of any of the aforementioned co-administered agents correspond to dosages currently used and can be reduced by the combined action (synergy) of the new agent and other chemotherapeutic agents or treatments.

[0287] Combination therapy can provide "synergy" and confirm the "synergistic effect", i.e. that the effect achieved with the combined use of active ingredients exceeds the sum of the effects obtained with the use of these compounds separately. A synergistic effect can be obtained if the active ingredients are: (1) co-formulated and administered or simultaneously delivered as part of a combined dosage formulation; (2) delivered sequentially or in parallel as separate formulations; or (3) through some other administration regimen. With sequential delivery, a synergistic effect can be obtained if the compounds are administered or delivered sequentially, for example, through different injections in separate syringes. In general, during sequential therapy, an effective dose of each active ingredient is administered sequentially, i. one after the other, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

IV. Finished products and kits

[0288] Another embodiment of the present disclosure is an article of manufacture containing materials useful in the treatment, prevention, and/or diagnosis of a proliferation disorder. In one embodiment, the finished product comprises a container and a label or package insert on or associated with it. Suitable containers include, for example, bottles, vials, syringes, etc. The containers can be formed from various materials such as glass or plastic. The container contains a composition effective for treating, preventing and/or diagnosing a cancerous condition and may have a sterile entry port (for example, the container may be an intravenous solution bag or a vial containing a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-CD79b antibody immunoconjugate of the present disclosure. The label or package insert indicates that the composition is used to treat cancer. The label or package insert will additionally contain instructions for administering the composition to a patient with cancer. Alternatively, the finished product may further comprise a second container containing a pharmaceutically 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 also contain other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

[0289] In some embodiments, the finished product is a container, such as a bottle, vial, syringe, that includes a pharmaceutical composition of the present disclosure. In some embodiments, the finished product is a container, such as a bottle, vial, syringe, that includes a lyophilized pharmaceutical composition (eg, mass) of the present disclosure. In some embodiments, the finished product is a container, such as a bottle, vial, syringe, that includes the reconstituted lyophilized pharmaceutical composition of the present disclosure. In a particular embodiment, the finished product is a container that includes a lyophilized pharmaceutical composition reconstituted with Sterile Water for Injection (SWFI).

[0290] In some embodiments, the finished product is a plastic container that includes the pharmaceutical composition of the present disclosure. In some embodiments, the finished product is a glass container that includes the pharmaceutical composition of the present disclosure.

[0291] In some embodiments, the finished product is a glass vial that includes the pharmaceutical composition of the present disclosure. In a particular embodiment, the finished product is a glass vial that includes a lyophilized pharmaceutical composition (such as a mass) of the present disclosure. In a particular embodiment, the product is a glass vial that includes a reconstituted lyophilized pharmaceutical composition (such as a bulk) of the present disclosure. In a particular embodiment, the finished product is a glass vial that includes a lyophilized pharmaceutical composition (eg, bulk) reconstituted with Sterile Water for Injection (SWFI).

In some embodiments, the finished product is an intravenous sachet containing the pharmaceutical composition or liquid composition of the present disclosure. An intravenous bag may be composed of materials including, but not limited to, polyolefin (PO), polyvinyl chloride (PVC), ethylene vinyl acetate, polypropylene (PP), polyethylene (PE), copolyester, or a combination thereof. In some embodiments, the IV bag is a polyolefin (PO) bag, a polypropylene (PP) bag, a polyethylene (PE) bag, or a polyvinyl chloride (PVC) bag.

[0292] In some embodiments, the finished product containing the pharmaceutical composition or liquid composition of the present disclosure may have 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 at least about 55 ml, at least about 60 ml, at least about 65 ml, at least about 70 ml, at least about 75 ml, at least about 80 ml, at least about 85 ml, at least about 90 ml, at least about 95 ml, at least about 100 ml, at least about 105 ml, at least about 110 ml, at least about 115 ml, at least about 120 ml, at least about 125 ml, at least about 130 ml, at least about 135 ml, at least about 140 ml, at least about 145 ml, at least about 150 ml, at least about 155 ml, at least about 160 ml, at least about 165 ml, at least about 170 ml, at least about 175 ml, 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 finished product is a bag of 110 that can hold a volume of at least about 25 ml, at least about 50 ml, or at least about 100 ml. In some embodiments, the finished product is a PVC bag that can hold a volume of at least about 25 ml, at least about 50 ml, or at least about 100 ml.

[0293] In some embodiments, the finished product is an intravenous pouch comprising a pharmaceutical composition or liquid composition of the present disclosure and a buffer. In a particular embodiment, the buffer may be saline, half saline, 5% w/v. dextrose, Ringer's lactate solution, or a combination thereof. In one embodiment, the finished product is an intravenous sachet comprising a pharmaceutical composition or liquid composition of the present disclosure dissolved in half saline.

[0294] In some embodiments, the finished product is an intravenous pouch comprising the pharmaceutical composition or liquid composition of the present disclosure and an isotonic buffer. In one embodiment, the finished product is an intravenous sachet comprising a pharmaceutical composition or liquid composition of the present disclosure dissolved in saline.

[0295] In one exemplary embodiment, the finished product is a PVC bag comprising the pharmaceutical composition or liquid composition of the present disclosure and a saline solution that can hold a volume of at least about 100 ml. In another exemplary embodiment, the finished product is a PVC bag comprising a pharmaceutical composition or liquid composition of the present disclosure and a saline solution that can hold a volume of about 100 ml.

[0296] In another exemplary embodiment, the finished product is a PO bag comprising a pharmaceutical composition or liquid composition of the present disclosure and a saline solution that can hold a volume of at least about 100 ml. In yet another exemplary embodiment, the finished product is a PO bag comprising a pharmaceutical composition or liquid composition of the present disclosure and a half saline solution that can hold a volume of at least about 100 ml.

[0297] In any of the embodiments of this invention, the stable pharmaceutical or liquid pharmaceutical composition may be stored in a container such as a bottle, vial, syringe, or intravenous (IV) bag.

[0298] Kits are also provided that can be used for a variety of purposes, such as kill assays for CD79b-expressing cells. The finished product contains a container and a label or package insert on or associated with it. The container contains a composition containing at least one immunoconjugate containing an anti-CD79b antibody of the present disclosure. Additional containers may be included which contain, for example, diluents, surfactants, buffers, control antibodies. The label or package insert may contain a description of the composition as well as instructions for the intended use.

[0299] The following description is presented to enable a person skilled in the art to create and apply various implementation options. The description of specific devices, methods, and applications is provided by way of example only. Various modifications to the examples described herein will be apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various implementations. Thus, the various embodiments are not intended to be limited to the examples described and shown, but are intended to fall within the scope of the claims.

EXAMPLES

Example 1 Surfactant Studies for Anti-CD79b Immunoconjugate

[0300] The formulation originally used for early phase clinical trials in anti-CD79b-vc-MMAE patients was a liquid formulation containing a histidine acetate buffer with 0.02% polysorbate 20 delivered to patients via an intravenous syringe pump. To develop a commercial drug formulation, various non-ionic surfactants were tested at various concentrations to determine if they could protect and stabilize anti-CD79b-vc-MMAE from aggregation when diluted in an IV bag, which could be by delivery to patients under commercial conditions. In particular, the stability of anti-CD79b-vc-MMAE was evaluated under both (a) static conditions in the IV bag and (b) air-water interfacial stress conditions caused by short-term agitation in the IV bag. .

Anti-CD79b-vc-MMAE

[0301] Anti-CD79b-vc-MMAE was diluted in 20 mM histidine acetate, pH 5.5. Buffer 20 mm histidine acetate pH 5.5 was prepared by adding 3.11 g of histidine and 1.03 ml of glacial acetic acid in 800 ml of fresh ultrapure water. The measured pH was 5.5±0.1. The resulting solution was made up to 1 L using fresh ultrapure water, filtered through a 0.22 μM PES filter, and stored at 2-8°C. Anti-CD79b-vc-MMAE concentrations were tested at both (i) 10 mg/mL and (ii) 20 mg/mL by dilution with filtered buffer to 10 mg/mL or 20 mg/mL as required. The diluted material was stored at 2-8°C protected from light.

Surface-active substance

[0302] The following surfactants were tested: (i) polysorbate 20 (PS20), USP, NF purity, manufactured by Croda; (ii) polysorbate 80 (PS80), USP, NF grade, manufactured by Croda; (iii) poloxamer (P188), USP, NF, containing 98 ppm butylated hydroxytoluene, manufactured by Spectrum Chemicals; and (iv) N-octyl-β-D glucopyranoside (OG) C14H2806, FW: 292.4, available from Affymetrix, Anagrade. Compositions of 10%, 1.0%, and 0.5% PS20 stock solution were prepared as follows: depending on the target PS20 concentration in the bag, 10%, 1%, or 0.5% PS20 stock solution in ultrapure water was used. A 10% stock solution of PS20 was prepared by adding 10 g of PS20 to a 100 ml volumetric flask. The flask was filled with 60 ml of ultrapure water and then stirred with a magnetic stirrer to mix the solution well. After the bubbles had subsided for 30 minutes, the flask was filled to 100 ml with ultrapure water. The solution was transferred to a light-protective container and stored for a maximum of 1 week at 2-8°C. A similar procedure was used to prepare a 1.0% PS20 solution using 1.0 g PS20 in 100 ml ultrapure water. To prepare a 0.5% PS20 solution, a 1:1 (v/v) dilution of a 1.0% PS20 stock solution was used.

[0303] The preparation of 0.5% PS80 was as follows: Stock 10% PS20 was prepared by adding 10 g PS80 to 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 allowed to stand for 30 minutes to allow the bubbles to subside and then stirred. The volume of the solution was then adjusted to 100 ml using ultrapure water. To obtain a 0.5% PS80 stock solution, 0.5 ml of a 10% PS80 stock solution was adjusted to 10 ml with ultrapure water. The solutions were transferred to a light-protective container and stored for a maximum of 1 week at 2-8°C.

[0304] The preparation of 0.5% P188 was as follows: Stock 10% P188 was prepared by adding 10 g of P188 to 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 allowed to stand for 30 minutes to allow the bubbles to subside and then stirred. The volume of the solution was then adjusted to 100 ml using ultrapure water. To obtain a 0.5% P188 stock solution, 0.5 ml of a 10% P188 stock solution was adjusted to 10 ml with ultrapure water. The solutions were transferred to a light-protective container and stored for a maximum of 1 week at 2-8°C.

A. Static I/O Packet Research:

[0305] Anti-CD79b-vc-MMAE was diluted in IV bags. To mimic the recommended pharmacy preparation procedure, the same volume of saline was removed from the IV bag as the total volume of anti-CD79b-vc-MMAE and surfactant added. Surfactants were injected into the IV bags prior to administration of the anti-CD79b-vc-MMAE composition in histidine buffer, ensuring that the anti-CD79b-vc-MMAE as a surfactant-free ultrafiltration diafiltration (UFDF) pool did not exposed to saline without any presence of surfactant in the IV bag. The IV bags were then gently swirled to ensure complete mixing while avoiding vigorous shaking and mixing in bags containing anti-CD79b-vc-MMAE.

[0306] Preparation of anti-CD79b-vc-MMAE in saline contained in glass vials: Varying amounts of saline (0.9% sodium chloride), surfactants and immunoconjugate were added to a 15 cc Forma Vitrum glass vial and capped Daikyo stopper with a diameter of 20 mm. Later, the samples were incubated under certain research conditions.

[0307] Sample collection: During the study, samples were collected using a 1 cc or 5 cc BD Falcon syringe in combination with an 18 G needle. Collected samples were stored in 10 cc PETG containers (Nalgene).

[0308] Determination of protein concentration by UV spectrophotometric scanning: Protein concentration after dilution was determined for selected samples using an Agilent spectrophotometer. Samples collected from IV bags were diluted (if necessary) by volume so that the UV signal was between 0.1 and 1.0 FU. Absorption was recorded at 279 nm and 320 nm. UV concentration determination was calculated using an extinction coefficient of 1.40 (mg/mL)-1 cm-1. The corrected A279 was obtained by subtracting A320 nm from A279 nm. This correction takes into account the turbidity of the solution and allows you to accurately measure the protein concentration.

[0309] Haze: The haze of the samples was measured by recording the average UV absorbance in the range of 340-360 nm using a cuvette with a path length of 1 cm in an Agilent spectrophotometer. The baseline sample for the spectrophotometer was purified water.

[0310] Visible particles: The presence of any visible particles was assessed visually against the black and white background of a light box with samples in 10 cc PETG containers. Soluble aggregate content as a measure of protein instability was determined using high performance size exclusion chromatography (E-HPLC) on an Agilent 1200/1280 (Agilent Technologies, Santa Clara, CA) equipped with a diode array detector set to 280 nm and Tosoh Bioscience LLC (Montgomeryville, PA) TSK-Gel G3000SWXL size-exclusion column (300 x 7.8 mm, 5 µm) at ambient temperature. Samples were eluted for 30 min at an isocratic flow rate of 0.5 ml/min using 0.2 M K3PO4, 0.25 M KCl, pH 6.2.

[0311] High Precision Liquid Particle Counter (HIAC): HIAC has been used to size and count particles in solution. Light dimming was used to quantify visible and/or invisible particles (SVP) using a HIAC instrument (Model 9703+). 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.

Stability studies in IV bags under static storage:

[0312] To investigate the effect of surfactants in IV bags, the initial study was conducted under both static and agitated conditions. The dose range tested in these studies for anti-CD79b-vc-MMAE was designed and performed in the range of 1.8 mg/kg to 2.4 mg/kg. Most patients were expected to weigh between 40 and 120 kg. Based on these assumptions, the total dose per IV bag ranged from 72 mg (low dose/low weight) to 288 mg (high dose/high weight).

[0313] FIG. 1-2 summarize the results for IV bags with a fixed PS20 concentration of 0.005% per bag and a final anti-CD79b-vc-MMAE concentration of 0.65 mg/mL. The concentration of anti-CD79b-vc-MMAE in the IV bag was selected based on a low dose of 1.8 mg/kg for the lowest patient weight of 40 kg, in a 100 ml IV bag. Since the 100 ml IV bags were overfilled by 10 ml, the final concentration in the IV bag was: (1.8 mg/kg × 40 kg)/110 ml = 0.65 mg/ml

[0314] Under static storage conditions at 30°C for 22 hours, there were no significant differences between the types of IV bags in terms of physical stability of anti-CD79b-vc-MMAE. All packages showed an increase (A) in the content of macromolecular compounds (HMC) by 0.3% + 0.1%, measured using SEC. This demonstrated that anti-CD79b-vc-MMAE was unstable at 30° C. when diluted in 0.9% NaCl.

Surfactant and screening studies in static storage:

[0315] For well-controlled and efficient screening of surfactant type and surfactant concentration, it was necessary to apply a fixed concentration of protein in the bag. The concentration of anti-CD79b-vc-MMAE in the 2.6 mg/mL pouch was chosen based on the maximum possible dose of 2.4 mg/kg for a 120 kg heavy patient administered in a 100 mL pouch, which is the recommended pouch size for clinic and which has a 10% overflow volume as seen from the following calculation: (2.4 mg/kg × 120 kg)/110 ml = 2.6 mg/ml. It was assumed that the highest concentration of protein in the bag would be the worst case, since higher volume concentrations of protein would cause faster adsorption of the antibody at the air-water interface, resulting in higher levels of interfacial mediated aggregation.

Effect of Temperature on Static Storage Stability:

[0316] Providing suitable storage conditions for the composite IV bag is important to the quality of the therapeutic antibody product, and this has been appreciated. This information is also required for the preparation of pharmacy manuals and product inserts to ensure that any therapeutic antibodies are used correctly. Various levels of PS20 in 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 pouch (0.003%, 0.005% and 0.01%) did not show any significant change in the number of IUDs after 22 hours of static storage (FIG. 3). All changes (% IUD) were <0.02%. At 25°C, and PS20 levels of 0.005%, 0.01%, 0.015%, 0.025%, and 0.030% per sachet, an increase of ~0.1% IUD was observed after 22 hours of static storage (Figure 3). The increase in IUD at 30°C was much greater than that observed at 25°C. At PS20 levels of 0.004%, 0.015%, 0.02%, 0.025%, 0.05% and 0.1% in the package after 22 hours of static storage, an increase in the number of IUDs by 0.3 + 0.1% was observed (Fig. 3 ).

[0317] At all temperatures, the increase in the number of IUDs was independent of the concentration of PS20 in the packet, indicating that aggregation under static conditions is not mediated by interfacial stress. Increasing the number of IUDs showed a strong correlation with storage temperature. These results correlate well with subsequent studies by Beckley, N. et al., Investigation into Temperature-Induced Aggregation of an Antibody Drug Conjugate. Bioconjugate Chemistry., 24, pp: 1674-1683 (2013), indicating that the formation of IUDs in antibody-drug conjugates (KALP) at higher temperatures is due to increased sensitivity of the tertiary structure to thermal stress, presumably due to the presence of conjugated drugs in interchain cysteines close to the CH2 domain.

Testing of surfactant blends:

[0318] Poloxamer 188 (P188) is known to reduce the formation of IUDs in IV bags, but a significant increase in the number of invisible particles (SVP) can be observed with mixing. In contrast, PS20 did not increase SVP. To further investigate these different results between the two surfactants, a mixture of PS20 - P188 in an equal 1:1 weight ratio was used to determine if PS20 could prevent SVP formation and P188 could limit the increase in IUDs. The study was conducted in 25 ml IV bags with an anti-CD79b-vc-MMAE concentration of 2.6 mg/ml per bag. On a weight basis, equal amounts of PS20 and P188 were added to give a total surfactant concentration in the IV bag of 0.05% or 0.1%. An increase in the number of IUDs of 0.3-0.4% was observed at static state at a temperature of 30°C for 22 hours in Table 1 below.

[0319] There was a slight increase in particle sizes over 5 µm, 10 µm and 25 µm. When 0.1% surfactant was used in the IV bag, an increase in 2 µm particles was observed. Based on the results, the surfactant blend showed no significant difference compared to PS20 alone.

[0320] Four different surfactants were evaluated in this study, including PS20, PS80, P188, and octylglucoside (OG). After incubation at 30°C for 22 hours with 0.02% surfactant and 2.6 mg/ml drug product in vials filled with saline from PVC bags, an increase in the amount of IUD by 0.3-0 was observed, 4%, demonstrating that aggregation was also independent of the type of surfactant used (FIG. 4).

Stability of anti-CD79b-vc-MMAE under static storage in vials:

[0321] Given the physical instability of anti-CD79b-vc-MMAE in saline (0.9% NaCl) when statically stored in an IV bag, the stability of the immunoconjugate in 5% dextrose (D5W) and half saline (0. 45% w/v NaCl) was tested in vials to assess the dependence of immunoconjugate instability on ionic strength. Solutions containing 0.02% PS20 were prepared with a fixed concentration of anti-CD79b-vc-MMAE of 2.6 mg/ml. Anti-CD79b-vc-MMAE showed a significant improvement in physical stability in both half saline and D5W (FIG. 4). In the D5W solution, there was no change in the number of IUDs, while in half saline, the number of IUDs increased by no more than 0.1% after 24 hours of static storage at 30°C. Although the drug in D5W limited anti-CD79b-vc-MMAE aggregation, antibody glycation in D5W solution is a known risk (see Fischer, S. et al., Glycation during Storage and Administration of Monoclonal Antibody Formulations. European J Pharma and Biopharm. vol.70;pp.42-50 (2008)). Thus, iv delivery of anti-CD79b-vc-MMAE with 5% dextrose sachets was not considered further due to the potential for glycation. Although half saline has shown a significant improvement in physical stability compared to conventional saline, due to the low availability of prepackaged half saline pouches in global clinical and commercial settings, as well as the inconvenience and potential risks of microbial contamination, related to the preparation of half saline, the use of half saline for intravenous infusion of anti-CD79b-vc-MMAE was also not preferred.

Conclusions of static stability studies in IV bags and glass vials:

[0322] The static stability studies described above demonstrate that anti-CD79b-vc-MMAE aggregation is driven by a high ionic strength environment, indicating that the rate of aggregation in formulation buffer is significantly lower than in saline.

B. Stirring studies in IV bags:

[0323] There have been a number of studies of agitation in IV bags to support transport of multiple IV bags containing anti-CD79b-vc-MMAE in a hospital or clinical setting for dosing. Although transportation of prepared IV bags containing therapeutic proteins is not generally recommended due to the risk of aggregation, there is often a practical need to transport IV bags in clinical settings, especially in remote locations with limited access to pharmacies. Therefore, a minimum volume of transport must be maintained, and the effect on the drug due to agitation of the IV bag must be evaluated during development. Stress in IV bags during transport can cause physical degradation of the therapeutic antibody. Physical degradation (ie, aggregation and particle formation) of the antibody is likely mediated by adsorption at the air-liquid interface in the IV bag, which undergoes continuous regeneration during agitation. In the studies presented below, a MaxQ 4000 elliptical laboratory shaker (orbital length 0.75 inches) was used for mixing studies in IV bags. IV bags were placed in the plane of the shaker.

Effect of Temperature on Agitation Stability in IV Pouches:

[0324] Stirring effects studies were conducted with different levels of PS20 in the bag at 2-8°C, 25°C and 30°C (FIG. 5A - FIG. 5C). The rate of increase in the number of IUDs was greater at 30°C than at 25°C, which was greater than at 2-8°C. The increase in the amount of IUD correlated with temperature during mixing in the IV bag. For packets that contain PS20 concentrations of 0.01%, 0.005%, and 0.003%, there was no increase in the amount of IUD when stirred for 2 hours at 2-8°C (FIG. 5A). At 30°C, the increase in the number of IUDs depended on the concentration of PS20 in the bag (Fig. 5C). It was noted that the increase in the amount of HMC with stirring at all concentrations of PS20 both at 2-8°C (Fig. 5A) and at 25°C was non-linear (Fig. 5B). This non-linear behavior is likely due to sampling at different time points.

Effect of surfactant blend on agitation stability in an IV bag:

[0325] A mixture of PS20 - P188 in an equal ratio by weight of 1:1 was used to determine if PS20 could prevent the formation of SVP, and P188 to limit the increase in the amount of HMC with mixing. The study was conducted in 25 ml IV bags with an anti-CD79b-vc-MMAE concentration of 2.6 mg/ml per bag. On a weight basis, equal amounts of PS20 and P188 were added to give a total surfactant concentration in the IV bag of 0.05% or 0.1%.

[0326] When 0.05% of the total surfactant was used in the IV bag, a 2.9% increase in IUD was observed after stirring at 30° C. for 2 hours (Table 2). There was a trend towards increasing particle sizes over 5 µm, 10 µm and 25 µm up to 1 hour, and the number of particles exceeded the limit of detection (LO) after 2 hours. When 0.1% of the total surfactant was used in the IV sachet, there was a tendency to increase the number of particles of all sizes, but this was significantly lower than in the 0.05% surfactant sachet (Table 2). The IUD increase was also lower by about 2.8% after 2 hours of mixing compared to 0.05% total surfactant concentration in the IV bag. Based on these results, the surfactant blend showed no difference compared to PS20 alone.

Brief mixing in an IV bag at 30°C:

[0327] The purpose of this study was to study the transport of IV bags with diluted anti-CD79b-vc-MMAE for a short time under environmental conditions and to determine the effective concentration of PS20 in the composition of anti-CD79b-vc-MMAAE. In this study, the package was subjected to short-term mixing at 30°C for 30 minutes. This shorter agitation at 30° C. may override the potential agitation that diluted anti-CD79b-vc-MMAE may experience during transport in a hospital or other facility under environmental conditions. The study was conducted with 0.001%, 0.002%, 0.003% and 0.005% PS20 diluted in an IV bag and mixed at 30°C and 100 rpm. The results are shown in FIG. 6. According to the results, at 0.005% PS20 in the package, there is an increase of <0.1%. Under these conditions, for anti-CD79b-vc-MMAE, there is a direct correlation of the increase in IUD with the concentration of PS20 in the IV bag. Based on this study, to ensure physical stability of the diluted anti-CD79b-vc-MMAE in the IV bag, the minimum amount of PS20 in the IV bag was between 0.003% and 0.005% to keep cumulative levels below the limit of quantitation .

Stirring at 2-8°C while cooling the IV bag (without pre-cooling the bags):

[0328] The aim of this study was to support the transport of composed packets for i/o in a more realistic scenario. Typically IV bags are formulated by pharmacists and transported in vehicles that may or may not be pre-refrigerated. Before starting this experiment, the packages were equilibrated to 30°C to simulate the higher ambient temperatures that may exist in different parts of the world. This ensures the stability of the diluted drug composition (DPC) even if the packages were not pre-chilled or transported in a container that was not pre-chilled. The study was performed with 2.6 mg/ml anti-CD79b-vc-MMAE with four different levels of PS20 in 100 ml IV bags: 0.001%, 0.002%, 0.003% and 0.005%. There is an increase in IUDs of <0.1% in IV bags with ≤0.002% PS20. Based on the results of this mixing model study, the minimum amount of PS20 in the IV bag ranged from 0.001% to 0.002% to keep cumulative levels below the limit of quantitation. The results are shown in FIG. 7.

Conclusions:

[0329] Antibody drug conjugates (KALP) are particularly more sensitive to saline and high ionic strength buffers than conventional unconjugated antibodies (Adem, Y., et al. Bioconjugate Chemistry, 25 (2014) 656-664). Tested anti-CD79b-vc-MMAE showed an increase in IUDs when diluted in 0.3% saline at 30° C. for 22 hours in static storage.

[0330] Based on two mixing models, anti-CD79b-vc-MMAE is stable at a content of at least 0.002% PS20 in the IV bag during mixing caused by short-term transportation for 1 hour at 2-8°C. To meet agitation stability requirements during handling, transport, and short term storage, the final suggested concentration of surfactant in the anti-CD79b-vc-MMAE formulation is 0.12%, resulting in 0.004% per pouch for the lowest expected patient weight (40 kg ) in combination with the use of a 100 ml IV bag. This level of surfactant has been shown to protect anti-CD79b-vc-MMAE over a dose range of 1.8 mg/kg to 2.4 mg/kg. It should be noted that this amount of surfactant, in particular PS20, is surprisingly high and counterintuitive in terms 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 for the anti-CD79b-vc-MMAE drug product is also unexpectedly higher compared to another immunoconjugate, anti-CD22-vc-MMAE, for which a commercial formulation with 0.05% PS20 was found to be suitable for reconstitution in sachets. for I/O (data not shown).

Example 2 Reduced Risks of Anti-CD79b Immunoconjugate Oxidation and Linker Hydrolysis

[0331] Establishing a concentration of surfactant suitable for IV bag delivery as described in Example 1 potentially exacerbates the problem of anti-CD79b-vc-MMAE oxidation. It is known that higher levels of polysorbate are generally associated with higher levels of peroxide due to auto-oxidative degradation of polysorbate. See, Lam HM, 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.

[0332] In addition, it is known that the oxidation of tryptophan 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. Notably, for the anti-CD79b antibody used in the present disclosure, tryptophan is present in the HVR 1 variable heavy chain: GYTFSSYWIE (SEQ ID NO: 4). Propensity to oxidize under AAPH stress conditions (e.g., 2 weeks at 40°C, 6 months at 25°C, or 6 months at 2-8°C) was determined by mass spectrometric analysis of tryptic peptides after anti-CD79b- vc-MMAE in AARN. Stressed anti-CD79b-vc-MMAEs were digested with trypsin and digested peptides were subjected to LC-MS/MS to determine percent oxidation (data not shown). Peptide mapping showed 68% oxidation of HVR tryptophan. This resulted in a loss of about 58% of anti-CD79b-vc-MMAE activity (data not shown). Therefore, it was necessary to reduce the risk of oxidation.

[0333] Additional factors to consider include how to manage succinimide hydrolysis of the immunoconjugate linker. The present CD79b-vc-MMAE immunoconjugate uses a maleimide-containing drug-linker conjugated to antibody cysteine residues to form thiosuccinimide bonds. It is known that such thiosuccinimide linkages can undergo two competing reactions in the patient's plasma: (a) maleimide elimination, resulting in undesirable loss of drug from the immunoconjugate; and (b) hydrolysis of the thiosuccinimide ring to form a succinic acid derivative which cannot be cleaved off. See Fig. 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 the potential to affect pharmacokinetics (PK) and safety. It has previously been demonstrated that the initial liquid formulation used for the early phase of clinical trials in patients, e.g., the liquid formulation in histidine acetate buffer at pH 5.5, showed an increase in acidity of about 9% under recommended storage conditions over 2 years (data not shown) .

[0334] As a result of these three risks: increased risk of oxidation due to elevated PS20 concentrations; increased risk of oxidation due to tryptophan oxidation, which can lead to loss of activity; and the risk of linker hydrolysis which could result in loss of MMAE drug from the immunoconjugate and result in the presence of free drug in the patient's plasma, a lyophilized formulation has been proposed to reduce these risks resulting in a longer and more stable shelf life.

A. Exploring Alternative Buffer Types:

[0335] To initiate formulation development for a lyophilized product, alternative buffer types have been explored. The buffer used in the composition at an early stage of the clinical phase was histidine acetate buffer. It is known that L-histidine can be oxidized 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, acetate can evaporate during the lyophilization process, resulting in a pH change that can destabilize the immunoconjugate. In addition, the presence of acetate can be considered as a potential hazard to the lyophilization process, since acetate is highly flammable. Therefore, alternative types of buffers were evaluated. Three compositions were tested as shown in

[0336] The stability of the lyophilized-thawed anti-CD79b-vc-MMAE pharmaceutical compositions in Table 3 in disposable PETG containers was evaluated under freeze/thaw stress conditions. The protein compositions were sterilized by filtration and filled into PETG containers. The containers were placed at -20°C for T=0 and 1 week. The containers were frozen and thawed a total of five times to room temperature. Only T = 0 and the third freeze-thaw cycle were analyzed. The results are shown in Table 4.

[0337] The results show that no problems were observed with any of the three compositions tested. Therefore, the use of histidine or succinate would be an acceptable alternative to histidine acetate.

[0338] A stability screen was then performed, checking the effect of pH on the formation of the charge variant at 30°C. Using the same three liquid compositions as in Table 3, which were stressed at 30° C. at three different time points of 2 weeks, 4 weeks and 8 weeks, the variant charge distribution was assessed using the iCE280 analyzer (ProteinSimple) with a PrinCE microinjector and fluorocarbon coated capillary cartridge 100 µm × 5 cm (ProteinSimple). To remove the lysine residue at the C-terminus of the heavy chain, carboxypeptidase B (CpB) was added to each sample after a dilution step at a ratio of enzyme to substrate of 1:1000 (wt:wt). In addition, sialidase A was added to remove sialic acid. After adding CpB and sialidase A, the samples were incubated at 37° C. for 10 minutes. Samples of the incubated liquid composition were mixed with an ampholyte solution consisting of a mixture of 700 µl of 1% methylcellulose, 1218 µl of purified water, 8 µl of farmalite 8-10.5, 55 µl of farmalite 5-8, 15 µl of pI marker 5, 12, 4 µl of pI marker 7.05. The samples were focused by applying a potential of 1500 V for 1 minute, then a potential of 3000 V for 5 minutes with 80 mM phosphoric acid anolyte and 100 mM sodium hydroxide catholyte, both in 0.1% methylcellulose. An image of focused charge variants was obtained by passing 280 nm ultraviolet (UV) light through a capillary into a digital camera lens of a CCD. The results are shown in FIG. 9 and FIG. 10, which showed that between pH 5.0 and 5.5, there was less formation of acid forms and slightly more formation of basic forms. Thus, it appears that the buffer components do not have a significant effect on charge stability.

B. Investigation of the effect of pH on stability:

[0339] Screening tests on the effect of pH on stability and on the formation of 30°C size variants were performed using the same three liquid formulations as in Table 3 that were stressed at 30°C at three different time points of 2 weeks, 4 weeks, and 8 weeks. Variant size distribution was determined by size exclusion chromatography (SEC) on an Agilent Technologies 1200 Series HPLC (Santa Clara, CA) using 0.25 mM potassium chloride, and a mobile phase of pH 6.2. Liquid composition samples were then loaded onto a Tosoh Bioscience TSKgel G3000SWXL column (South San Francisco, CA). Samples were eluted over 30 minutes using a flow rate of 0.5 ml/min and absorbance was monitored at 280 nm. The results are presented as relative peak area versus total area under the curve and are shown in FIG. 11. Stability screening showed that between pH 5.0 and 5.5 there were fewer macromolecular compounds (HMCs) compared to pH 6.0. Similar amounts of low molecular weight compounds (NMS) were observed after 4 weeks at 30°C. After 8 weeks, slightly higher NMS values were observed at 30°C. Since lowering the pH to 5.3 appears to reduce both charge and size formation, a pH of 5.3 was chosen.

[0340] In FIG. 12 shows a chromatogram testing two types of buffer: histidine/histidine-HCl at pH 5.5 and sodium succinate at three different pH: 5.0, 5.5 and 6.0. Histidine buffer at pH 5.5 showed slightly better physical stability in liquid at 30°C than sodium succinate at any pH tested (pH 5.0, 5.5 or 6.0). However, because sodium succinate buffer exhibited acceptable fluid stability, and given the potential risk of histidine oxidation, sodium succinate buffer was chosen.

C. Testing of lyophilized compositions:

[0341] The lyophilized composition was tested to reduce the risks described above. The general scheme for the lyophilization cycle is presented in Table 5.

[0342] The duration of the cycle was about 78 hours or about 3 days. The moisture content ranged from 0.5% to 0.7%.

[0343] A lyophilization screening study was performed at different concentrations of anti-CD79B-VC-MMAE, two different types of buffer, different pH, and two different concentrations of surfactants, as shown in Table 6.

[0344] These lyophilized test formulations were stored at 30° C. for up to 2 months as part of a screening study.

[0345] To quantify any monomers developed for each test composition (1) - (5) as an indicator of stability, EC analysis was used after reconstitution of each test composition to 10 mg / ml or to 20 mg / ml, as described above . Fig. 13 summarizes this study and demonstrates that during 2 months of heat stress at 30°C, there was no change in the percentage of monomers in any of the compositions tested. Each lyophilized composition (1) - (5) was also tested using vkIEF, as described above, to determine the percentage of the main peak after 2 months of heat stress at 30°C. 14 summarizes this study and demonstrates that during 2 months of heat stress at 30°C, there was no change in the percentage of the main peak in any of the tested compositions. As a result of these two studies, it was determined that sodium succinate buffer in the pH range of 5.0 to 6.0 resulted in acceptable stability of the tested lyophilized compositions.

D. Appearance of the lyophilized mass:

[0346] An important aspect of lyophilized compositions is the actual appearance of the lyophilized mass, since it is an indication of the quality of the product. For the anti-CD79b-vc-MMAE freeze-dried mass, both immunoconjugate concentration and stabilizer/tonizer concentration were tested to explore how to improve the appearance of the freeze-dried mass. On FIG. 15A - Fig. 15C depicts three lyophilized compositions with a different protein at the sucrose ratio tested. Indentations were visible at the bottom of the lyophilized mass for all three levels of sucrose tested, which did not create an ideal appearance of the lyophilized mass. However, under similar lyophilization conditions, formulations with less sucrose show smaller depressions, and lower concentrations of sucrose result in lower 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. On FIG. 16A and FIG. 16B is a comparison of the appearance of two lyophilized masses. The concentration of immunoconjugate 20 mg/ml in combination with 120 mm sucrose showed the most stable and uniform appearance of the lyophilized mass. When conducting heat stress resistance studies for 4 weeks at 40°C, 25°C and 5°C, no change in the structure, color or appearance of the mass was noticed, and the moisture content remained <5%.

E. Stability of the lyophilized composition:

[0347] Lyophilized drug formulation: 20 mg/mL anti-CD79b-vc-MMAE, 120 mM sucrose, 0.12% PS20 in 10 mM sodium succinate at pH 5.3 tested for stability as measured by formation of soluble aggregate or variant charge. Soluble aggregates were measured by SEC as described above. On FIG. 17 illustrates that for the lyophilized drug product, there was less than a 0.1% increase in soluble aggregates at 25°C and 2-8°C for 8 weeks. At 40°C, an increase in the number of dimeric aggregates of about 0.1% per week was observed, and the rate of this aggregation may depend on the level of humidity. The charge variants were measured using the ICEF as described above. On FIG. 18 illustrates that for the lyophilized drug product, there was a loss of about 5% in the main peak, and an increase of 3.7% in the acid version after 4 weeks at 40°C. There were no significant changes at 25°C and 2-8°C. As a result, no changes were observed for the lyophilized drug product under stress conditions at 25°C and 2-8°C.

[0348] Table 7 summarizes the properties for the final anti-CD79B-VC-MMAE formulation from the multiple studies described in Examples 1 and 2.

[0349] 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 results in improved bulk appearance when the solution is lyophilized. 0.12% PS20 (ie 1.2 mg/mL) protects the protein in the formulation from interfacial stresses that occur during administration using IV bags and IV infusion sets. The pH value of 5.3 minimizes the formation of acid variants produced by the hydrolysis of succinimide. The composition in Table 7 is designed to deliver 140 mg drug product per vial after reconstitution in 7.2 ml CVID to maintain a clinical dose of 1.8 mg/kg.

Example 3: Evaluation of drug substance resistance and anti-CD79b-vc-MMAE drug products

[0350] It was found that the drug product (see Table 7) in the lyophilized composition is stable for at least 44 months at 2°C - 8°C in the range of residual moisture from 0.3% (wt./mass.) up to 3.2% (w/w) and also for at least 7 months at 25°C in the range of residual moisture from 0.3% (w/w) to 3.2% (w/w) masses).

[0351] Then, the composition parameters of the lyophilized drug product were varied and evaluated for a practically significant effect on the stability of the lyophilized drug product. Five formulation parameters were tested (and the ranges in which the parameters varied): (a) protein concentration (17-23 mg/ml), (b) succinate concentration (7-23 mM), (c) sucrose concentration (90-150 mM), (d) polysorbate 20 concentration (0.9-1.5 mg/ml), and (e) pH (4.95-5.65). There was no practically significant effect on the stability of the lyophilized drug product after storage at 2-8° C. for up to nine months. Based on the degradation rates predicted by the models, no practically significant effect on the stability of the lyophilized drug product is expected after storage at 2-8°C for at least 24 months.

[0352] The reconstituted drug solution (e.g., 20 mg/mL polatuzumab vedotin in 10 mM succinate, 120 mM sucrose, 1.2 mg/mL polysorbate 20, pH 5.3) was physicochemically stable after 72 hours of storage at 2°C - 8°C or after 24 hours storage at 30°C with ambient light. There were no significant changes (eg, statistically significant changes) in the affinity of polatuzumab vedotin for its target (i.e., CD79b) or in the biological activity of polatuzumab vedotin.

[0353] Although the foregoing invention has been described in detail by way of illustration and examples for the purposes of clarity of understanding, the descriptions and examples should not be taken as limiting the scope of the present invention. Disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety and expressly.

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SEQUENCE LIST

<110>Genentech, Inc.

<120> STABLE IMMUNOCONJUGATE COMPOSITIONS CONTAINING ANTIBODIES TO CD79B

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<---

Claims (84)

1. A stable liquid composition for administering an immunoconjugate containing an anti-CD79b antibody, comprising an immunoconjugate containing an anti-CD79b antibody, a buffering agent, a sugar, and a surfactant, wherein the concentration of the immunoconjugate containing the antibody to CD79b is from 5 mg/ml to 60 mg/ml; the concentration of the buffer agent is from 10 mm to 200 mm; the sugar concentration is from 100 mM to 260 mM; the surfactant is polysorbate 20 at a concentration of at least 1.2 mg/ml, where the pH of the liquid composition is in the range from 5.0 to 6.0, and while the immunoconjugate containing the antibody to CD79b has the formula: , Where Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, while the light chain contains (a) the HVR-L1 sequence KASQSVDYEGDSFLN (SEQ ID NO: 1); (b) HVR-L2 AASNLES sequence (SEQ ID NO: 2); And (c) HVR-L3 sequence QQSNEDPLT (SEQ ID NO: 3); while the heavy chain contains (a) HVR-H1 GYTFSSYWIE sequence (SEQ ID NO: 4); (b) the HVR-H2 sequence GEILPGGGDTNYNEIFKG (SEQ ID NO: 5); And (c) the sequence of HVR-H3 TRRVPIRLDY (SEQ ID NO: 6); Val is valine; Cit is citrulline; and p has a value from 1 to 8. 2. A stable liquid composition for administering an immunoconjugate containing an anti-CD79b antibody according to claim 1, characterized in that the concentration of an immunoconjugate containing an anti-CD79b antibody is from 10 mg/ml to 20 mg/ml, optionally the concentration of an immunoconjugate containing an anti-CD79b antibody. CD79b is 20 mg/ml. 3. A stable liquid composition for administering an immunoconjugate containing an anti-CD79b antibody according to claim 1 or 2, characterized in that the buffering agent is a histidine buffer or a succinate buffer, optionally the succinate buffer is a sodium succinate buffer. 4. A stable liquid composition for administering an immunoconjugate containing an anti-CD79b antibody according to claim 3, characterized in that the concentration of sodium succinate buffer is 10 mM. 5. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-4, characterized in that the liquid composition has a pH of 5.3. 6. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-5, characterized in that the sugar is selected from the group consisting of: sucrose, mannitol, sorbitol, glycerol, dextran 40 and trehalose, optionally the sugar is sucrose. 7. A stable liquid composition for administering an immunoconjugate containing an anti-CD79b antibody according to claim 6, characterized in that the sugar concentration is 120 mM. 8. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-7, characterized in that the concentration of polysorbate 20 is 1.2 mg/ml. 9. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-8, characterized in that the concentration of the immunoconjugate containing the antibody to CD79b is 20 mg / ml, the buffer agent is a sodium succinate buffer at a concentration of 10 mm, the surfactant is polysorbate 20 at a concentration of 1.2 mg / ml, and the sugar is sucrose at a concentration of 120 mm, while the liquid composition has a pH of 5.3. 10. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-9, characterized in that the liquid composition was recovered from the lyophilized composition. 11. A stable liquid formulation for administering an anti-CD79b immunoconjugate according to claim 10, characterized in that the liquid formulation was reconstituted using Sterile Water for Injection (SWFI), optionally the liquid composition was reconstituted using 7.2 ml of SFI. 12. Stable lyophilized composition for the administration of an immunoconjugate containing an antibody to CD79b, containing 150 mg of an immunoconjugate containing an antibody to CD79b, 9.0 mg of polysorbate 20, 8.88 mg of succinic acid, 4.08 mg of sodium hydroxide and 309 mg of sucrose, moreover an immunoconjugate containing an anti-CD79b antibody has the formula: , Where Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, while the heavy chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 10; Val is valine; Cit is citrulline; and p has a value between 2 and 5. 13. Stable lyophilized composition for the administration of an immunoconjugate containing an antibody to CD79b, containing 140 mg of an immunoconjugate containing an antibody to CD79b, 8.4 mg of polysorbate 20, 8.27 mg of succinic acid, 3.80 mg of sodium hydroxide and 288 mg of sucrose, and an immunoconjugate containing an anti-CD79b antibody has the formula: , Where Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, while the heavy chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 10; Val is valine; Cit is citrulline; and p has a value between 2 and 5. 14. Stable lyophilized composition for the administration of an immunoconjugate containing an antibody to CD79b, containing 30 mg of an immunoconjugate containing an antibody to CD79b, 1.8 mg of polysorbate 20, 1.77 mg of succinic acid, 0.816 mg of sodium hydroxide and 61.8 mg of sucrose, moreover an immunoconjugate containing an anti-CD79b antibody has the formula: , Where Ab is an anti-CD79b antibody, wherein the anti-CD79b antibody contains a heavy chain and a light chain, while the heavy chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 9 and the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO: 10; Val is valine; Cit is citrulline; and p has a value between 2 and 5. 15. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 10, 11, characterized in that the lyophilized composition is a lyophilized mass. 16. Stable lyophilized composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 12-14, characterized in that the lyophilized composition is a lyophilized mass. 17. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, obtained by restoring the lyophilized composition according to any one of paragraphs. 12-14 and 16. 18. A stable liquid composition for administering an immunoconjugate containing an anti-CD79b antibody according to claim 17, characterized in that the lyophilized composition is reconstituted with sterile water for injection (SWFI), optionally the lyophilized composition is reconstituted with 7.2 ml of SWFI. 19. A stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, containing a) 0.72-2.7 mg/ml of an immunoconjugate containing an antibody to CD79b; b) 0.36 mM-1.35 mM sodium succinate; c) 0.51 mM-16.24 mM sucrose; d) 0.0432 mg/ml-0.162 mg/ml polysorbate 20, while the pH of the liquid composition is from 5 to 5.7, and while the immunoconjugate containing the antibody to CD79b is polatuzumab vedotin. 20. A stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to claim 19, containing a) 0.72 mg/ml immunoconjugate containing anti-CD79b antibody; b) 0.36 mM sodium succinate; c) 0.51 mM sucrose; And d) 0.0432 mg/ml polysorbate 20, wherein the pH of the liquid composition is between 5.1 and 5.4, optionally the pH of the liquid composition is 5.3. 21. A stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to claim 19, containing a) 2.7 mg/ml immunoconjugate containing anti-CD79b antibody; b) 1.35 mM sodium succinate; c) 16.24 mM sucrose; And d) 0.162 mg/ml polysorbate 20, wherein the pH of the liquid composition is between 5.1 and 5.4, optionally the pH of the liquid composition is 5.3. 22. Stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 19-21, characterized in that the ratio of the immunoconjugate containing the antibody to CD79b to polysorbate 20 in the composition is 0.72 mg/ml: 0.0432 mg/ml. 23. A stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, obtained by dissolving a liquid composition for the administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-11, 15 and 17, 18 in isotonic buffer. 24. A stable liquid composition for intravenous administration of an immunoconjugate containing an anti-CD79b antibody according to claim 23, characterized in that the concentration of the surfactant after dilution is at least 0.004% wt./about. 25. Stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 19-24, characterized in that the volume of the liquid composition is 50-100 ml, optionally the volume of the liquid composition is 50 ml or 100 ml. 26. Stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 19-25, characterized in that the liquid composition is in a bag for intravenous (IV) administration. 27. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-11 and 15, characterized in that a) the anti-CD79b antibody contains 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; or b) the heavy chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO:9 and the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO:10. 28. Stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 23-26, characterized in that a) the anti-CD79b antibody contains 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; or b) the heavy chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO:9 and the light chain of the anti-CD79b antibody contains the amino acid sequence of SEQ ID NO:10. 29. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-11, 15, 17, 18 and 27, characterized in that p has a value from 2 to 5, optionally p has a value of about 3.5. 30. Stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-11, 15, 17, 18, 27 and 29, characterized in that the immunoconjugate containing the anti-CD79b antibody is polatuzumab vedotin. 31. Stable lyophilized composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 12-14 and 16, characterized in that p has a value of about 3.5. 32. Stable lyophilized composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 12-14, 16 and 31, characterized in that the immunoconjugate containing the antibody to CD79b is polatuzumab vedotin. 33. Stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 23-26 and 28, characterized in that p is between 2 and 5, optionally p is about 3.5. 34. Stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 23-26, 28 and 33, characterized in that the immunoconjugate containing the anti-CD79b antibody is polatuzumab vedotin. 35. The use of a stable liquid composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 1-11, 15, 17, 18, 27 and 29, 30 in the manufacture of a medicament for the treatment of a B cell proliferative disorder in a patient in need thereof, wherein the B cell proliferation disorder is selected from the group consisting of: lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), relapsed/refractory forms of diffuse large B-cell lymphoma, 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 cell lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma. 36. The use of a stable lyophilized composition for the introduction of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 12-14, 16 and 31, 32 in the manufacture of a medicament for the treatment of a B cell proliferative disorder in a patient in need thereof, wherein the B cell proliferation disorder is selected from the group consisting of: lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), relapsed/refractory forms of diffuse large B-cell lymphoma, 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 cell lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma. 37. The use of a stable liquid composition for intravenous administration of an immunoconjugate containing an antibody to CD79b, according to any one of paragraphs. 19-26, 28 and 33, 34 in the manufacture of a medicament for the treatment of a B cell proliferative disorder in a patient in need thereof, wherein the B cell proliferation disorder is selected from the group consisting of: lymphoma, myeloma, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), relapsed/refractory forms of diffuse large B-cell lymphoma, 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 cell lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.