Antibody formulations

Abstract

Provided herein are pharmaceutical compositions comprising: an anti-ROR1 antibody or a ROR1 binding antibody fragment thereof, and about 5% (w / v) or thicker trehalose; wherein the anti-ROR1 antibody or ROR1 thereof binds to an antibody fragment. The pharmaceutical compositions disclosed herein reduce the formation of aggregates and maintain high purity of antibodies or antigen-binding fragments thereof.

Description

[0001] Cross - reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 367,195, filed Jun. 28, 2022, which is incorporated herein by reference.

[0003] Incorporation by reference of sequence listing

[0004] This application is being filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled 51956725_601_SL.xml, created on Jun. 22, 2023, and having a size of 10871 bytes. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Background of the Invention

[0005] Cancer is one of the leading causes of death worldwide. In the United States alone, cancer causes more than half a million deaths each year, and approximately 1.4 million new cases are diagnosed annually. Receptor tyrosine kinases (RTKs) play key roles in cell differentiation, proliferation, migration, angiogenesis, and survival. Receptor tyrosine kinase - like orphan receptor 1 (ROR1) is a type I membrane protein belonging to the ROR subfamily, and its extracellular domain contains immunoglobulin (Ig) - like domains, a cysteine - rich domain, and a kringle domain. Recent research progress has revealed the possible role of ROR1 in the non - canonical WNT signaling pathway to promote the survival of malignant cells. Studies have also shown that the non - canonical WNT signaling pathway plays an important role in the metastasis of basal - like carcinomas and other subtypes of cancer. Developing and mass - producing antibodies against ROR1 to a level suitable for clinical applications is an important step in developing ROR1 - targeted therapies. Summary of the Invention

[0006] Over the past few decades, the use of antibodies as drugs has been increasing. In many cases, most therapeutic antibodies are injected or infused via the intravenous (IV) route. The challenge in developing and scaling up therapeutic antibodies is that, typically, the amount of antibody that can be administered via the intravenous route is limited by physicochemical properties. For example, the amount of antibody is determined by its solubility and stability in a suitable liquid formulation and the volume of the infusion fluid. Regardless of the route, the injection route usually ultimately requires injecting a solution containing a high concentration of protein. One problem to be solved is how to develop a pharmaceutical formulation of highly concentrated, stable, and pharmaceutically active ROR1 antibodies or mixtures of such antibody molecules for injection (e.g., intravenous injection or infusion).

[0007] Compositions for pharmaceutical formulations useful for generating highly concentrated, stable, pharmaceutically active antibodies and antigen - binding antibody fragments are provided herein.

[0008] A composition comprising: (a) an antibody or antigen-binding fragment thereof at a concentration of about 20 - 80 mg / mL; (b) a buffer; (c) a chelating agent; (d) a surfactant; and trehalose dihydrate at a concentration of 5.0% (w / v) or higher; wherein the pH of the composition is 6.0 or lower. In some embodiments, the antibody is a ROR1 antibody. In some embodiments, the antibody is zilovertamab. In some embodiments, the antibody buffer comprises acetate, citrate, histidine, arginine, succinate, or phosphate. In some embodiments, the buffer of the antibody comprises sodium citrate. In some embodiments, the concentration of the antibody buffer in the composition is 5 mM to 100 mM. In some embodiments, the concentration of the antibody buffer in the composition is 5 mM to 50 mM. In some embodiments, the concentration of the antibody buffer in the composition is 5 mM to 20 mM. In some embodiments, the concentration of the buffer of the antibody in the composition is about 10 mM. In some embodiments, the chelating agent of the antibody comprises ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). In some embodiments, the chelating agent of the antibody comprises EDTA. In some embodiments, the concentration of the chelating agent of the antibody in the composition is about 0.01 mM to 0.2 mM. In some embodiments, the concentration of the chelating agent of the antibody in the composition is about 0.01 mM to 0.1 mM. In some embodiments, the concentration of the chelating agent of the antibody in the composition is about 0.02 mM to 0.2 mM. In some embodiments, the concentration of the chelating agent of the antibody in the composition is about 0.05 mM. In some embodiments, the antibody surfactant comprises polysorbate 80, polysorbate 20, or a polyethylene-polypropylene copolymer. In some embodiments, the antibody surfactant comprises polysorbate 80. In some embodiments, the concentration of the antibody surfactant present in the composition is about 0.005% (w / v) to 0.1% (w / v). In some embodiments, the concentration of the antibody surfactant present in the composition is about 0.01% (w / v) to 0.05% (w / v). In some embodiments, the concentration of the antibody surfactant present in the composition is about 0.01% (w / v) to 0.03% (w / v). In some embodiments, the concentration of the antibody surfactant present in the composition is about 0.02% (w / v). In some embodiments, the concentration of the antibody trehalose dihydrate present in the composition is about 5.0% (w / v) to 10.0% (w / v). In some embodiments, the concentration of the antibody trehalose dihydrate present in the composition is about 6.0% (w / v) to 9.0% (w / v).In some embodiments, the antibody trehalose dihydrate is present in the composition at a concentration of about 7.0% (w / v) to 8.0% (w / v). In some embodiments, the antibody trehalose dihydrate is present in the composition at a concentration of about 7.5% (w / v). In some embodiments, the pH of the antibody is about 4.0 to 6.0. In some embodiments, the pH of the antibody is about 4.5 to 6.0. In some embodiments, the pH of the antibody is about 4.0 to 5.5. In some embodiments, the pH of the antibody is about 4.5 to 5.5. In some embodiments, the pH of the antibody is about 5.0 to 5.5. In some embodiments, the pH of the antibody is about 5.2. In some embodiments, the antibody composition is liquid. In some embodiments, a method of treating cancer is described herein, comprising administering the composition to an individual in need thereof. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the leukemia or lymphoma is B-cell leukemia or lymphoma. In some embodiments, the leukemia or lymphoma is chronic lymphocytic leukemia (CLL). In some embodiments, the leukemia or lymphoma is mantle cell lymphoma (MCL). In some embodiments, the cancer is solid tissue cancer. In some embodiments, the solid tissue cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, head and neck cancer, kidney cancer, colon cancer, or gastric cancer. In some embodiments, the composition reduces the formation of aggregates. In some embodiments, the composition contains less than 2% antibody aggregates. In some embodiments, the aggregates include precipitates, and the composition reduces the formation of precipitates. In some embodiments, the composition contains less than 2% antibody aggregates after incubation at 40°C for 4 weeks. In some embodiments, the composition maintains the purity of the antibody or its antigen-binding fragment. In some embodiments, the composition maintains the purity of the antibody or its antigen-binding fragment at at least 97%. In some embodiments, the composition maintains the purity of the antibody or its antigen-binding fragment at at least 97% after incubation at 40°C for 4 weeks. In some embodiments, the buffer concentration in the composition is up to 10 mM. In some embodiments, the buffer is sodium citrate.

[0009] Also described herein is a composition comprising: (a) an antibody or its antigen-binding fragment at a concentration of about 40 milligrams per milliliter; (b) about 10 mM sodium citrate; (c) about 0.05 mM EDTA; (d) about 0.02% (w / v) polysorbate 80; and (e) about 7.5% (w / v) trehalose dihydrate; wherein the pH of the composition is about 5.2. In some embodiments, the antibody is a ROR1 antibody. In some embodiments, the antibody is zilovertamab.

[0010] The present disclosure also describes a composition comprising: (i) an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 20 to about 80 milligrams per milliliter (mg / mL); and (ii) trehalose at a concentration of about 5% (w / v) or higher; wherein the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises: (a) a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence shown in SEQ ID NO:1; (b) a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid sequence shown in SEQ ID NO:2; (c) a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid sequence shown in SEQ ID NO:3; (d) a light chain complementarity determining region 1 (L-CDR1) comprising the amino acid sequence shown in SEQ ID NO:4; (e) a light chain complementarity determining region 2 (L-CDR2) comprising the amino acid sequence shown in SEQ ID NO:5; and / or (f) a light chain complementarity determining region 3 (L-CDR3) comprising the amino acid sequence shown in SEQ ID NO:6; wherein the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof binds to ROR1. In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises: (a) a heavy chain variable domain having 90% or higher sequence identity with SEQ ID NO:7; and (b) a light chain variable domain having 90% or higher sequence identity with SEQ ID NO:8. In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises: (a) a heavy chain variable domain comprising SEQ ID NO:7; and (b) a light chain variable domain comprising SEQ ID NO:8. In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises: (a) a heavy chain constant domain having 90% or higher sequence identity with SEQ ID NO:11. In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises: (a) a heavy chain domain having 90% or higher sequence identity with SEQ ID NO:9; and (b) a light chain domain having 90% or higher sequence identity with SEQ ID NO:10. In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises: (a) a heavy chain domain comprising SEQ ID NO:9; and (b) a light chain variable domain comprising SEQ ID NO:10. In some embodiments, the anti-ROR1 antibody is zilovertamab. In some embodiments, the concentration of the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof is about 30 mg / mL to about 50 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof is about 40 mg / mL.In some embodiments, the composition comprises about 7% (w / v) or more trehalose. In some embodiments, the composition comprises about 7.5% (w / v) trehalose. In some embodiments, the composition further comprises a citrate buffer. In some embodiments, the composition comprises less than about 20 mM citrate buffer. In some embodiments, the composition comprises less than about 15 mM citrate buffer. In some embodiments, the composition comprises about 5 mM to about 20 mM citrate buffer. In some embodiments, the composition comprises about 10 mM citrate buffer. In some embodiments, the composition is buffered to a pH of about 4.5 to 5.5. In some embodiments, the composition further comprises a nonionic surfactant. In some embodiments, the nonionic surfactant is polysorbate 80. In some embodiments, the composition comprises about 0.010% (w / v) to about 0.050% (w / v) polysorbate 80. In some embodiments, the composition further comprises a chelating agent. In some embodiments, the chelating agent comprises EDTA. In some embodiments, the composition comprises: (a) about 40 mg / mL anti-ROR1 antibody or an ROR1-binding antibody fragment thereof; (b) about 7.5% (w / v) trehalose dihydrate; (c) about 10 mM citrate buffer; (d) about 0.02% (w / v) polysorbate 80; and (e) about 0.05 mM EDTA. In some embodiments, the composition reduces aggregate formation. In some embodiments, the aggregates comprise precipitate, and the composition reduces precipitate formation. In some embodiments, the composition comprises less than 2% antibody aggregates. In some embodiments, the composition comprises less than 2% antibody aggregates after incubation at 40 °C for 4 weeks. In some embodiments, the composition maintains the purity of the antibody or its antigen-binding fragment. In some embodiments, the composition maintains at least 97% purity of the antibody or its antigen-binding fragment. In some embodiments, the composition maintains at least 97% purity of the antibody or its antigen-binding fragment after incubation at 40 °C for 4 weeks. In some embodiments, the buffer concentration in the composition is up to 10 mM. In some embodiments, the buffer is sodium citrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the present disclosure are set forth in detail in the appended claims. The features and advantages of the present disclosure may be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0012] Figure 1 SEC-MALS chromatogram data for an anti-ROR1 antibody formulation.

[0013] Figure 2 Data showing increased thermal stability and reduced antibody aggregate formation of an anti-ROR1 antibody preparation. Detailed Description

[0014] Anti-ROR1 antibody

[0015] Provided herein are stable antibody preparations, as well as antibodies and antibody fragments that bind to ROR1 in the form of stable preparations. For example, provided herein are anti-ROR1 antibodies that, in certain cases, inhibit cancer cell growth and metastasis. In some embodiments, an anti-ROR1 antibody composition can be used in a method of inhibiting metastasis using an anti-ROR1 antibody or an antibody fragment thereof. In some embodiments, the antibody binds to an Ig-like domain adjacent to the CRD domain of human ROR-1 (hROR1). In some embodiments, the anti-ROR1 antibody binds to an epitope mapped to amino acids 42-160 of hROR-1. In some embodiments, the anti-ROR1 antibody binds to an epitope mapped to amino acids 130-160 of hROR-1.

[0016] The stable pharmaceutical preparations of the anti-ROR1 antibodies or ROR1-binding antibody fragments thereof provided herein include:

[0017] (a) Heavy chain complementarity-determining region 1 (H-CDR1) that comprises the amino acid sequence shown in SEQ ID NO:1;

[0018] (b) Heavy chain complementarity-determining region 2 (H-CDR2) that comprises the amino acid sequence shown in SEQ ID NO:2;

[0019] (c) Heavy chain complementarity-determining region 3 (H-CDR3) that comprises the amino acid sequence shown in SEQ ID NO:3;

[0020] (d) Light chain complementarity-determining region 1 (L-CDR1) that comprises the amino acid sequence shown in SEQ ID NO:4;

[0021] (e) Light chain complementarity-determining region 2 (L-CDR2) that comprises the amino acid sequence shown in SEQ ID NO:5; and / or

[0022] (f) Light chain complementarity-determining region 3 (L-CDR3) that comprises the amino acid sequence shown in SEQ ID NO:6.

[0023] Further provided are stable pharmaceutical preparations of an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof, which comprise:

[0024] (a) Heavy chain complementarity-determining region 1 (H-CDR1);

[0025] (b) Heavy chain complementarity-determining region 2 (H-CDR2);

[0026] (c) Heavy chain complementarity-determining region 3 (H-CDR3);

[0027] (d) Light chain complementarity-determining region 1 (L-CDR1);

[0028] (e) Light chain complementarity-determining region 2 (L-CDR2); and / or

[0029] (f) Light chain complementarity-determining region 3 (L-CDR3); wherein H-CDR1, H-CDR2, and H-CDR3 are derived from SEQ ID NO:7, and L-CDR1, L-CDR2, and L-CDR3 are derived from SEQ ID NO:8; wherein H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 are defined using the Chothia, Kabat, IMGT, Contact, or AbM method.

[0030] In some embodiments, the anti-ROR1 antibody or its ROR1-binding antibody fragment comprises:

[0031] A heavy chain variable domain comprising an amino acid sequence having 70% or higher sequence identity to SEQ ID NO:7; and

[0032] A light chain variable domain comprising an amino acid sequence having 70% or higher sequence identity to SEQ ID NO:8.

[0033] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 80% or higher sequence identity to SEQ ID NO:7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 85% or higher sequence identity to SEQ ID NO:7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 90% or higher sequence identity to SEQ ID NO:7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 95% or higher sequence identity to SEQ ID NO:7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 98% or higher sequence identity to SEQ ID NO:7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 99% or higher sequence identity to SEQ ID NO:7.

[0034] In some embodiments, the light chain variable domain comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO:8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO:8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO:8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO:8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 98% or higher sequence identity with SEQ ID NO:8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 99% or higher sequence identity with SEQ ID NO:8.

[0035] In some embodiments, the anti-ROR1 antibody or its ROR1-binding antibody fragment comprises:

[0036] a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:7; and

[0037] a light chain variable domain comprising the amino acids shown in SEQ ID NO:8.

[0038] In some embodiments, the anti-ROR1 antibody or its ROR1-binding antibody fragment comprises a heavy chain constant domain that comprises a sequence having 70% or higher sequence identity with SEQ ID NO:11 in amino acids. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO:11. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO:11. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO:11. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO:11. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having 98% or higher sequence identity with SEQ ID NO:11. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having 99% or higher sequence identity with SEQ ID NO:11. In some embodiments, the anti-ROR1 antibody or its ROR1-binding antibody fragment comprises a heavy chain constant domain that comprises the amino acid sequence shown in SEQ ID NO:11.

[0039] In some embodiments, the anti-ROR1 antibody comprises:

[0040] a heavy chain domain comprising an amino acid sequence having 70% or higher sequence identity with SEQ ID NO:9; and

[0041] a light chain domain comprising an amino acid sequence having 70% or higher sequence identity with SEQ ID NO:10.

[0042] In some embodiments, the heavy chain domain comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO:9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO:9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO:9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO:9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 98% or higher sequence identity with SEQ ID NO:9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 99% or higher sequence identity with SEQ ID NO:9.

[0043] In some embodiments, the light chain domain comprises an amino acid sequence having 80% or higher sequence identity with SEQ ID NO:10. In some embodiments, the light chain domain comprises an amino acid sequence having 85% or higher sequence identity with SEQ ID NO:10. In some embodiments, the light chain domain comprises an amino acid sequence having 90% or higher sequence identity with SEQ ID NO:10. In some embodiments, the light chain domain comprises an amino acid sequence having 95% or higher sequence identity with SEQ ID NO:10. In some embodiments, the light chain domain comprises an amino acid sequence having 98% or higher sequence identity with SEQ ID NO:10. In some embodiments, the light chain domain comprises an amino acid sequence having 99% or higher sequence identity with SEQ ID NO:10.

[0044] In some embodiments, the anti-ROR1 antibody comprises:

[0045] a heavy chain domain comprising the amino acid sequence as shown in SEQ ID NO:9; and

[0046] a light chain domain comprising the amino acids as shown in SEQ ID NO:10.

[0047] In some embodiments, the anti-ROR1 antibody is zilovertamab (previously known as cirmtuzumab).

[0048] Pharmaceutical formulation

[0049] Provided herein are pharmaceutical compositions comprising the anti-ROR1 antibodies described herein. A composition or pharmaceutical formulation or pharmaceutical composition generally includes and / or refers to a preparation in a form such that the biological activity of the active ingredient contained therein can be effectively exerted and does not contain additional ingredients that are unacceptably toxic to the subject to whom the preparation is administered. A pharmaceutical formulation is generally sterile (e.g., aseptic or free of all live microorganisms and their spores). The pharmaceutical compositions described and provided herein generally comprise, in some embodiments, an anti-ROR1 antibody or an ROR1-binding antibody fragment mixed with one or more of the following: buffers, chelating agents, surfactants, and stabilizers (e.g., tonicity or osmolarity regulators).

[0050] Anti-ROR1 antibody

[0051] The pharmaceutical compositions described and provided herein comprise the anti-ROR1 antibodies or ROR1-binding antibody fragments described herein. In some embodiments, the concentration of the anti-ROR1 antibody or ROR1-binding antibody fragment in the composition is between 20 and 80 milligrams (mg) per milliliter (mL). In some embodiments, the concentration of the anti-ROR1 antibody or ROR1-binding antibody fragment in the composition is between 30 and 60 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody or ROR1-binding antibody fragment in the composition is between 25 and 50 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody or ROR1-binding antibody fragment in the composition is between 30 and 50 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody or ROR1-binding antibody fragment in the composition is between 35 and 45 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody or ROR1-binding antibody fragment in the composition is between 40 and 50 mg / mL.

[0052] In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment, present in the composition at a concentration of from about 20 mg / mL to about 80 mg / mL. In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment, present in the composition at a concentration of from about 20 mg / mL to about 80 mg / mL. In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment is present in the composition at a concentration of from about 20 mg / mL to about 25 mg / mL, from about 20 mg / mL to about 30 mg / mL, from about 20 mg / mL to about 35 mg / mL, from about 20 mg / mL to about 40 mg / mL, from about 20 mg / mL to about 45 mg / mL, from about 20 mg / mL to about 50 mg / mL, from about 20 mg / mL to about 55 mg / mL, from about 20 mg / mL to about 60 mg / mL, from about 20 mg / mL to about 65 mg / mL, from about 20 mg / mL to about 70 mg / mL, from about 20 mg / mL to about 80 mg / mL, from about 25 mg / mL to about 30 mg / mL, from about 25 mg / mL to about 35 mg / mL, from about 25 mg / mL to about 40 mg / mL, from about 25 mg / mL to about 45 mg / mL, from about 25 mg / mL to about 50 mg / mL, from about 25 mg / mL to about 55 mg / mL, from about 25 mg / mL to about 60 mg / mL, from about 25 mg / mL to about 65 mg / mL, from about 25 mg / mL to about 70 mg / mL, from about 25 mg / mL to about 80 mg / mL, from about 30 mg / mL to about 35 mg / mL, from about 30 mg / mL to about 40 mg / mL, from about 30 mg / mL to about 45 mg / mL, from about 30 mg / mL to about 50 mg / mL, from about 30 mg / mL to about 55 mg / mL, from about 30 mg / mL to about 60 mg / mL, from about 30 mg / mL to about 65 mg / mL, from about 30 mg / mL to about 70 mg / mL, from about 30 mg / mL to about 80 mg / mL, from about 35 mg / mL to about 40 mg / mL, from about 35 mg / mL to about 45 mg / mL, from about 35 mg / mL to about 50 mg / mL, from about 35 mg / mL to about 55 mg / mL, from about 35 mg / mL to about 60 mg / mL, from about 35 mg / mL to about 65 mg / mL, from about 35 mg / mL to about 70 mg / mL, from about 35 mg / mL to about 80 mg / mL, from about 40 mg / mL to about 45 mg / mL, from about 40 mg / mL to about 50 mg / mL, from about 40 mg / mL to about 55 mg / mL, from about 40 mg / mL to about 60 mg / mL, from about 40 mg / mL to about 65 mg / mL, from about 40 mg / mL to about 70 mg / mL, from about 40 mg / mL to about 80 mg / mL, from about 45 mg / mL to about 50 mg / mL,from about 45 mg / mL to about 55 mg / mL, from about 45 mg / mL to about 60 mg / mL, from about 45 mg / mL to about 65 mg / mL, from about 45 mg / mL to about 70 mg / mL, from about 45 mg / mL to about 80 mg / mL, from about 50 mg / mL to about 55 mg / mL, from about 50 mg / mL to about 60 mg / mL, from about 50 mg / mL to about 65 mg / mL, from about 50 mg / mL to about 70 mg / mL, from about 50 mg / mL to about 80 mg / mL, from about 55 mg / mL to about 60 mg / mL, from about 55 mg / mL to about 65 mg / mL, from about 55 mg / mL to about 70 mg / mL, from about 55 mg / mL to about 80 mg / mL, from about 60 mg / mL to about 65 mg / mL, from about 60 mg / mL to about 70 mg / mL, from about 60 mg / mL to about 80 mg / mL, from about 65 mg / mL to about 70 mg / mL, from about 65 mg / mL to about 80 mg / mL, or from about 70 mg / mL to about 80 mg / mL. In some embodiments, the anti-ROR1 antibody or ROR1-binding antibody fragment is present in the composition at a concentration of 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, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, or about 80 mg / mL.

[0053] Buffer

[0054] The present invention provides a pharmaceutical composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment and a buffer. A buffer generally comprises and / or refers to a reagent that maintains the pH of the solution of the preparation of the present invention within an acceptable range through the action of its acid / base conjugate components. Suitable pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, arginine buffer, gluconate buffer, succinate buffer, acetate buffer, acid buffer, glutamate buffer, other organic acid buffers, tromethamine buffer, and phosphate buffer.

[0055] In some embodiments, the buffer comprises citrate buffer. In some embodiments, the citrate buffer is sodium citrate. In some embodiments, the buffer comprises arginine buffer. In some embodiments, the buffer comprises gluconate buffer. In some embodiments, the buffer comprises succinate buffer. In some embodiments, the buffer comprises acetate buffer. In some embodiments, the buffer comprises lactate buffer. In some embodiments, the buffer comprises glutamate buffer. In some embodiments, the buffer comprises tromethamine buffer. In some embodiments, the buffer comprises phosphate buffer.

[0056] In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 20 mM (0.020 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 15 mM (0.015 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 25 mM (0.025 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 30 mM (0.030 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 25 mM. In some embodiments, the concentration of the buffering agent in the composition is from about 5 mM to about 10 mM, from about 5 mM to about 15 mM, from about 5 mM to about 20 mM, from about 5 mM to about 25 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 25 mM, from about 15 mM to about 20 mM, from about 15 mM to about 25 mM, or from about 20 mM to about 25 mM. In some embodiments, the concentration of the buffering agent in the composition is at most about 10 mM, about 15 mM, about 20 mM, or about 25 mM.

[0057] In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 50 mM (0.050 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 15 mM (0.015 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 55 mM (0.055 M). In some embodiments, the buffering agent is present in the composition at a concentration of from about 5 mM (0.005 M) to about 60 mM (0.060 M). In some embodiments, the concentration of the buffering agent in the composition is from about 5 mM to about 10 mM, from about 5 mM to about 15 mM, from about 5 mM to about 20 mM, from about 5 mM to about 50 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 50 mM, from about 15 mM to about 20 mM, from about 15 mM to about 50 mM, or from about 20 mM to about 50 mM. In some embodiments, the concentration of the buffering agent in the composition is at most about 10 mM, about 15 mM, about 20 mM, or about 50 mM. In some embodiments, the concentration of the buffer in the composition is up to 10 mM. In some embodiments, the buffer is sodium citrate.

[0058] In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 20 mM (0.020 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 15 mM (0.015 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 25 mM (0.025 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 30 mM (0.030 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM to about 25 mM. In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM to about 25 mM. In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM to about 10 mM, from about 5 mM to about 15 mM, from about 5 mM to about 20 mM, from about 5 mM to about 25 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 25 mM, from about 15 mM to about 20 mM, from about 15 mM to about 25 mM, or from about 20 mM to about 25 mM. In some embodiments, the concentration of the citrate buffer (e.g., sodium citrate) in the composition is at most about 10 mM, about 15 mM, about 20 mM, or about 25 mM. In some embodiments, the buffer concentration in the composition is up to 10 mM. In some embodiments, the buffer is sodium citrate.

[0059] In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 50 mM (0.050 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 15 mM (0.015 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 55 mM (0.055 M). In some embodiments, the citrate buffer (e.g., sodium citrate) is present in the composition at a concentration of from about 5 mM (0.005 M) to about 60 mM (0.030 M). In some embodiments, the concentration of the citrate buffer (e.g., sodium citrate) in the composition is from about 5 mM to about 10 mM, from about 5 mM to about 15 mM, from about 5 mM to about 20 mM, from about 5 mM to about 50 mM, from about 10 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 50 mM, from about 15 mM to about 20 mM, from about 15 mM to about 50 mM, or from about 20 mM to about 50 mM. In some embodiments, the concentration of the citrate buffer (e.g., sodium citrate) in the composition is at most about 10 mM, about 15 mM, about 20 mM, or about 50 mM. In some embodiments, the buffer concentration in the composition is up to 10 mM. In some embodiments, the buffer is sodium citrate.

[0060] In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 5 mM citrate buffer to about 25 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 5 mM citrate buffer to about 10 mM citrate buffer, from about 5 mM citrate buffer to about 20 mM citrate buffer, from about 5 mM citrate buffer to about 25 mM citrate buffer, from about 10 mM citrate buffer to about 20 mM citrate buffer, from about 10 mM citrate buffer to about 25 mM citrate buffer, or from about 20 mM citrate buffer to about 25 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises about 5 mM citrate buffer, about 10 mM citrate buffer, about 20 mM citrate buffer, or about 25 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises at least about 5 mM citrate buffer, about 10 mM citrate buffer, or about 20 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises at most about 10 mM citrate buffer, about 20 mM citrate buffer, or about 25 mM citrate buffer. In some embodiments, the buffer is present in the composition at a concentration of up to 10 mM, the buffer is sodium citrate, and the reduced concentration of up to 10 mM citrate buffer contributes to enhanced stability of the antibody formulation.

[0061] In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 5 mM citrate buffer to about 60 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 5 mM citrate buffer to about 10 mM citrate buffer, from about 5 mM citrate buffer to about 20 mM citrate buffer, from about 5 mM citrate buffer to about 30 mM citrate buffer, from about 5 mM citrate buffer to about 40 mM citrate buffer, from about 5 mM citrate buffer to about 50 mM citrate buffer, from about 5 mM citrate buffer to about 60 mM citrate buffer, from about 10 mM citrate buffer to about 20 mM citrate buffer, from about 10 mM citrate buffer to about 30 mM citrate buffer, from about 10 mM citrate buffer to about 40 mM citrate buffer, from about 10 mM citrate buffer to about 50 mM citrate buffer, from about 10 mM citrate buffer to about 60 mM citrate buffer, from about 20 mM citrate buffer to about 30 mM citrate buffer, from about 20 mM citrate buffer to about 40 mM citrate buffer, from about 20 mM citrate buffer to about 50 mM citrate buffer, from about 20 mM citrate buffer to about 60 mM citrate buffer, from about 30 mM citrate buffer to about 40 mM citrate buffer, from about 30 mM citrate buffer to about 50 mM citrate buffer, from about 30 mM citrate buffer to about 60 mM citrate buffer, from about 40 mM citrate buffer to about 50 mM citrate buffer, from about 40 mM citrate buffer to about 60 mM citrate buffer, or from about 50 mM citrate buffer to about 60 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises about 5 mM citrate buffer, about 10 mM citrate buffer, about 20 mM citrate buffer, about 30 mM citrate buffer, about 40 mM citrate buffer, about 50 mM citrate buffer, or about 60 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises at least about 5 mM citrate buffer, about 10 mM citrate buffer, about 20 mM citrate buffer, about 30 mM citrate buffer, about 40 mM citrate buffer, or about 50 mM citrate buffer. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises at most about 10 mM citrate buffer, about 20 mM citrate buffer, about 30 mM citrate buffer, about 40 mM citrate buffer, about 50 mM citrate buffer, or about 60 mM citrate buffer.

[0062] In some embodiments, the pH of a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment is from about 4.0 to about 6.0. In some embodiments, the pH of a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment is from about 4.5 to about 6.0. In some embodiments, the pH of a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment is from about 5.0 to about 6.0. In some embodiments, the pH of a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment is from about 4.5 to about 5.5. In some embodiments, the pH of a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment is from about 5.0 to about 5.5. In some embodiments, the pH of a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment is about 5.2.

[0063] Sugar

[0064] Provided herein are pharmaceutical compositions comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment and a sugar. Sugars or saccharides generally include and / or refer to molecules having the general composition (CH2O) n and their derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc.

[0065] The sugar can act as a stabilizer and / or a tonicity / osmolarity regulator. Stabilizers generally include and / or refer to pharmaceutically acceptable excipients that protect the active pharmaceutical ingredient (e.g., antibody) and / or the formulation from chemical and / or physical degradation during manufacture, storage, and application. Tonicity and / or osmolarity regulators generally include and / or refer to pharmaceutically acceptable excipients that regulate the tonicity of the formulation.

[0066] In some cases, using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), increases the stability of anti-ROR1 antibodies or ROR1-binding antibody fragments. In some cases, using a higher concentration of trehalose (e.g., 5% or higher) also enables the use of a higher concentration of anti-ROR1 antibodies or ROR1-binding antibody fragments. In some other cases, using a higher concentration of trehalose (e.g., 5% or higher) also allows for the use of a lower concentration of buffer (e.g., citrate buffer). In some embodiments, the sugar is trehalose (e.g., trehalose dihydrate). In some embodiments, the concentration of trehalose in the composition is about 5% (w / v) or higher. In some embodiments, the concentration of trehalose in the composition is about 6% (w / v) or higher. In some embodiments, the concentration of trehalose in the composition is about 7% (w / v) or higher. In some embodiments, the concentration of trehalose in the composition is about 8% (w / v) or higher. In some embodiments, the concentration of trehalose in the composition is about 9% (w / v) or higher. In some embodiments, the concentration of trehalose in the composition is about 10% (w / v) or higher. For example, the percentage (%) weight / volume (w / v) can refer to the concentration in the form of trehalose or trehalose hydrate (e.g., trehalose-2H 2 O). Such w / v percentage concentrations herein are calculated based on the dihydrate, but also refer to an equivalent amount of trehalose anhydrate, monohydrate, trihydrate, etc., unless otherwise stated. In some embodiments, using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), reduces aggregate formation. In some embodiments, the aggregates include precipitates, and using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), reduces precipitate formation. In some embodiments, using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), reduces aggregate formation after incubation at 40 °C for 4 weeks. In some embodiments, using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), maintains the purity of the antibody or its antigen-binding fragment in the formulation. In some embodiments, using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), maintains the purity of the antibody or its antigen-binding fragment in the formulation under thermal stress. In some embodiments, using a higher concentration of non-reducing sugar, such as trehalose (e.g., 5% or higher), maintains the purity of the antibody or its antigen-binding fragment in the formulation at 97% after incubation at 40 °C for 4 weeks.

[0067] In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 5% (w / v) trehalose to about 12% (w / v) trehalose. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 5% (w / v) trehalose to about 6% (w / v) trehalose, from about 5% (w / v) trehalose to about 7% (w / v) trehalose, from about 5% (w / v) trehalose to about 8% (w / v) trehalose, from about 5% (w / v) trehalose to about 9% (w / v) trehalose, from about 5% (w / v) trehalose to about 10% (w / v) trehalose, from about 5% (w / v) trehalose to about 11% (w / v) trehalose, from about 5% (w / v) trehalose to about 12% (w / v) trehalose, from about 6% (w / v) trehalose to about 7% (w / v) trehalose, from about 6% (w / v) trehalose to about 8% (w / v) trehalose, from about 6% (w / v) trehalose to about 9% (w / v) trehalose, from about 6% (w / v) trehalose to about 10% (w / v) trehalose, from about 6% (w / v) trehalose to about 11% (w / v) trehalose, from about 6% (w / v) trehalose to about 12% (w / v) trehalose, from about 7% (w / v) trehalose to about 8% (w / v) trehalose, from about 7% (w / v) trehalose to about 9% (w / v) trehalose, from about 7% (w / v) trehalose to about 10% (w / v) trehalose, from about 7% (w / v) trehalose to about 11% (w / v) trehalose, from about 7% (w / v) trehalose to about 12% (w / v) trehalose, from about 8% (w / v) trehalose to about 9% (w / v) trehalose, from about 8% (w / v) trehalose to about 10% (w / v) trehalose, from about 8% (w / v) trehalose to about 11% (w / v) trehalose, from about 8% (w / v) trehalose to about 12% (w / v) trehalose, from about 9% (w / v) trehalose to about 10% (w / v) trehalose, from about 9% (w / v) trehalose to about 11% (w / v) trehalose, from about 9% (w / v) trehalose to about 12% (w / v) trehalose, from about 10% (w / v) trehalose to about 11% (w / v) trehalose, from about 10% (w / v) trehalose to about 12% (w / v) trehalose, or from about 11% (w / v) trehalose to about 12% (w / v) trehalose. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises about 5% (w / v) trehalose, about 6% (w / v) trehalose, about 7% (w / v) trehalose, about 8% (w / v) trehalose, about 9% (w / v) trehalose, about 10% (w / v) trehalose, about 11% (w / v) trehalose, or about 12% (w / v) trehalose.In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises at least about 5% (w / v) trehalose, about 6% (w / v) trehalose, about 7% (w / v) trehalose, about 8% (w / v) trehalose, about 9% (w / v) trehalose, about 10% (w / v) trehalose, or about 11% (w / v) trehalose. Such w / v percentage concentrations herein are calculated based on the dihydrate, but also refer to equivalent amounts of trehalose anhydrate, monohydrate, trihydrate, etc., unless otherwise specified.

[0068] Additionally, provided herein are formulations comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment and a non-reducing sugar (such as trehalose), wherein the non-reducing sugar is present in the composition at a concentration of at least 0.5% (w / v), at least 1% (w / v), at least 2% (w / v), at least 3% (w / v), or at least 4% (w / v). Such % w / v concentrations herein are calculated according to the dihydrate, but also refer to equivalent amounts of trehalosan, monohydrate, trihydrate, etc., unless otherwise specified.

[0069] Surfactant

[0070] Provided herein are pharmaceutical compositions comprising an anti-ROR1 antibody or an ROR1 antibody-binding fragment and a surfactant (e.g., one or more surfactants). Surfactants generally comprise and / or refer to pharmaceutically acceptable surfactants. In some embodiments, surfactants are typically added to formulations to provide stability, reduce and / or prevent aggregation, or prevent and / or inhibit protein damage during processing conditions (such as purification, filtration, lyophilization, transportation, storage, and delivery). In some embodiments of the present invention, surfactants can be used to provide additional stability to the active ingredient (such as an antibody). Examples of pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylated sorbitan fatty acid esters, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers, polyoxyethylene-polyoxypropylene copolymers, and sodium dodecyl sulfate (SDS). Suitable polyoxyethylene-sorbitol fatty acid esters include polysorbate 20 (e.g., polyoxyethylene sorbitol monolaurate) and polysorbate 80 (e.g., polyoxyethylene sorbitol monooleate).

[0071] In some embodiments, the surfactant comprises a nonionic surfactant. In some embodiments, the nonionic surfactant is present in the composition at a concentration of from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the nonionic surfactant is present in the composition at a concentration of from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the nonionic surfactant is present in the composition at a concentration of from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the nonionic surfactant is present in the composition at a concentration of from about 0.005% (w / v) to about 0.01% (w / v), from about 0.005% (w / v) to about 0.02% (w / v), from about 0.005% (w / v) to about 0.05% (w / v), from about 0.005% (w / v) to about 0.06% (w / v), from about 0.005% (w / v) to about 0.08% (w / v), from about 0.005% (w / v) to about 0.1% (w / v), from about 0.01% (w / v) to about 0.02% (w / v), from about 0.01% (w / v) to about 0.05% (w / v), from about 0.01% (w / v) to about 0.06% (w / v), from about 0.01% (w / v) to about 0.08% (w / v), from about 0.01% (w / v) to about 0.1 (w / v), from about 0.02% (w / v) to about 0.05% (w / v), from about 0.02% (w / v) to about 0.06% (w / v), from about 0.02% (w / v) to about 0.08% (w / v), from about 0.02% (w / v) to about 0.1% (w / v), from about 0.05% (w / v) to about 0.06% (w / v), from about 0.05% (w / v) to about 0.08% (w / v), from about 0.05% (w / v) to about 0.1% (w / v), from about 0.06% (w / v) to about 0.08% (w / v), from about 0.06% (w / v) to about 0.1% (w / v), or from about 0.08% (w / v) to about 0.1% (w / v). In some embodiments, the nonionic surfactant is present in the composition at a concentration of about 0.005% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.08% (w / v) or about 0.1% (w / v). In some embodiments, the nonionic surfactant is present in the composition at a concentration of at least about 0.005% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.05% (w / v), about 0.06% (w / v) or about 0.08% (w / v).In some embodiments, the nonionic surfactant is present in the composition at a concentration of about up to about 0.01% (w / v), about 0.02% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.08% (w / v), or about 0.1% (w / v).

[0072] In some embodiments, the surfactant comprises polysorbate 80. In some embodiments, the surfactant comprises polysorbate 80. In some embodiments, the concentration of polysorbate 80 present in the composition is from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 present in the composition is from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 present in the composition is from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 present in the composition is from about 0.005% (w / v) to about 0.01% (w / v), from about 0.005% (w / v) to about 0.02% (w / v), from about 0.005% (w / v) to about 0.05% (w / v), from about 0.005% (w / v) to about 0.06% (w / v), from about 0.005% (w / v) to about 0.08% (w / v), from about 0.005% (w / v) to about 0.1% (w / v), from about 0.01% (w / v) to about 0.02% (w / v), from about 0.01% (w / v) to about 0.05% (w / v), from about 0.01% (w / v) to about 0.06% (w / v), from about 0.01% (w / v) to about 0.08% (w / v), from about 0.01% (w / v) to about 0.1 (w / v), from about 0.02% (w / v) to about 0.05% (w / v), from about 0.02% (w / v) to about 0.06% (w / v), from about 0.02% (w / v) to about 0.08% (w / v), from about 0.02% (w / v) to about 0.1% (w / v), from about 0.05% (w / v) to about 0.06% (w / v), from about 0.05% (w / v) to about 0.08% (w / v), from about 0.05% (w / v) to about 0.1% (w / v), from about 0.06% (w / v) to about 0.08% (w / v), from about 0.06% (w / v) to about 0.1% (w / v), or from about 0.08% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 present in the composition is about 0.005% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.08% (w / v) or about 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 present in the composition is at least about 0.005% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.05% (w / v), about 0.06% (w / v) or about 0.08% (w / v).In some embodiments, polysorbate 80 is present in the composition at a concentration of about at most about 0.01% (w / v), about 0.02% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.08% (w / v), or about 0.1% (w / v).

[0073] Chelating agent

[0074] The present disclosure provides pharmaceutical compositions comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment and a chelating agent. A chelating agent generally comprises and / or refers to a moiety or group on a molecule that binds to a metal ion through one or more donor atoms. Chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA).

[0075] In some embodiments, the chelating agent is present in the composition at a concentration of about 0.001 mM to about 0.350 mM. In some embodiments, the chelating agent is present in the composition at a concentration of about 0.010 mM to about 0.100 mM. In some embodiments, the chelating agent is present in the composition at a concentration of about 0.020 mM to about 0.080 mM. In some embodiments, the chelating agent is present in the composition at a concentration of about 0.040 mM to about 0.060 mM. In some embodiments, the chelating agent is present in the composition at a concentration of about 0.050 mM.

[0076] In some embodiments, the chelating agent is EDTA. In some embodiments, EDTA is present in the composition at a concentration of about 0.001 mM to about 0.350 mM. In some embodiments, the concentration of EDTA in the composition is about 0.010 mM to about 0.100 mM. In some embodiments, the concentration of EDTA in the composition is about 0.020 mM to about 0.080 mM. In some embodiments, the concentration of EDTA in the composition is about 0.040 mM to about 0.060 mM. In some embodiments, the concentration of EDTA in the composition is about 0.050 mM.

[0077] In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 0.001 mM EDTA to about 0.350 mM EDTA. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 0.010 mM EDTA to about 0.100 mM EDTA. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 0.020 mM EDTA to about 0.080 mM EDTA. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises from about 0.040 mM EDTA to about 0.060 mM EDTA. In some embodiments, a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment comprises about 0.050 mM EDTA.

[0078] Composition

[0079] Provided herein are compositions (e.g., pharmaceutical compositions) that comprise:

[0080] an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof; and about 5% (w / v) or more trehalose;

[0081] wherein the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises:

[0082] (a) a heavy chain complementarity determining region 1 (H-CDR1) that comprises the amino acid sequence set forth in SEQ ID NO:1;

[0083] (b) a heavy chain complementarity determining region 2 (H-CDR2) that comprises the amino acid sequence set forth in SEQ ID NO:2;

[0084] (c) a heavy chain complementarity determining region 3 (H-CDR3) that comprises the amino acid sequence set forth in SEQ ID NO:3;

[0085] (d) a light chain complementarity determining region 1 (L-CDR1) that comprises the amino acid sequence set forth in SEQ ID NO:4;

[0086] (e) a light chain complementarity determining region 2 (L-CDR2) that comprises the amino acid sequence set forth in SEQ ID NO:5; and / or

[0087] (f) a light chain complementarity determining region 3 (L-CDR3) that comprises the amino acid sequence set forth in SEQ ID NO:6.

[0088] Further provided are compositions (e.g., pharmaceutical compositions) that comprise:

[0089] An anti-ROR1 antibody or an ROR1-binding antibody fragment thereof; and about 5% (w / v) or more trehalose;

[0090] wherein the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises:

[0091] (a) Heavy chain complementarity determining region 1 (H-CDR1);

[0092] (b) Heavy chain complementarity determining region 2 (H-CDR2);

[0093] (c) Heavy chain complementarity determining region 3 (H-CDR3);

[0094] (d) Light chain complementarity determining region 1 (L-CDR1);

[0095] (e) Light chain complementarity determining region 2 (L-CDR2); and / or

[0096] (f) Light chain complementarity determining region 3 (L-CDR3); wherein H-CDR1, H-CDR2, and H-CDR3 are derived from SEQ ID NO:7, and L-CDR1, L-CDR2, and L-CDR3 are derived from SEQ ID NO:8; wherein H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 are defined using the Chothia, Kabat, IMGT, Contact, or AbM method.

[0097] In some embodiments, the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises:

[0098] A heavy chain variable domain comprising an amino acid sequence having 70% or higher sequence identity with SEQ ID NO:7; and

[0099] A light chain variable domain comprising an amino acid sequence having 70% or higher sequence identity with SEQ ID NO:8.

[0100] In some embodiments, the anti-ROR1 antibody comprises:

[0101] A heavy chain domain comprising an amino acid sequence having 70% or greater sequence identity with SEQ ID NO:9; and

[0102] A light chain domain comprising an amino acid sequence having 70% or higher sequence identity with SEQ ID NO:10.

[0103] In some embodiments, the anti-ROR1 antibody comprises: A heavy chain constant domain comprising an amino acid sequence having 70% or higher sequence identity with SEQ ID NO:11.

[0104] Further provided is a composition (e.g., a pharmaceutical composition) comprising:

[0105] an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof; and about 5% (w / v) or more trehalose;

[0106] wherein the anti-ROR1 antibody or the ROR1-binding antibody fragment thereof comprises:

[0107] a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:7; and

[0108] a light chain variable domain comprising the amino acids shown in SEQ ID NO:8.

[0109] Provided herein is a composition (e.g., a pharmaceutical composition) comprising:

[0110] an anti-ROR1 antibody; and

[0111] trehalose at a concentration of about 5% (w / v) or higher;

[0112] wherein the anti-ROR1 antibody comprises:

[0113] a heavy chain domain comprising the amino acid sequence shown in SEQ ID NO:9; and

[0114] a light chain domain comprising the amino acids shown in SEQ ID NO:10.

[0115] Also provided is a composition (e.g., a pharmaceutical composition) comprising zilovertamab and 5% (w / v) or more trehalose;

[0116] In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 20 mg / mL to about 80 mg / mL. In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 20 mg / mL to about 60 mg / mL. In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 30 mg / mL to about 50 mg / mL. In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 40 mg / mL to about 50 mg / mL. In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 20 mg / mL to about 80 mg / mL. In some embodiments, the composition comprises an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof at a concentration of about 40 mg / mL.

[0117] In some embodiments, the composition comprises about 6% (w / v) or more trehalose. In some embodiments, the composition comprises about 7% (w / v) or more trehalose. In some embodiments, the composition comprises about 8% (w / v) or more trehalose. In some embodiments, the composition comprises about 9% (w / v) or more trehalose. In some embodiments, using a higher concentration of a non-reducing sugar, such as trehalose (e.g., 5, 6, 7, 8, 9% or more), can reduce aggregate formation. In some embodiments, the aggregates include precipitates, and using a higher concentration of a non-reducing sugar, such as trehalose (e.g., 5, 6, 7, 8, 9% w / v or more), can reduce precipitate formation. In some embodiments, using a higher concentration of a non-reducing sugar, such as trehalose (e.g., 5, 6, 7, 8, 9% or more), can reduce aggregate formation after incubation at 40 °C for 4 weeks. In some embodiments, using a higher concentration of a non-reducing sugar, such as trehalose (e.g., 5, 6, 7, 8, 9% w / v or higher), can maintain the purity of the antibody or its antigen-binding fragment in the formulation. In some embodiments, using a higher concentration of a non-reducing sugar, such as trehalose (e.g., 5, 6, 7, 8, 9% w / v or higher), can maintain the purity of the antibody or its antigen-binding fragment within the formulation under heat stress. In some embodiments, using a higher concentration of a non-reducing sugar, such as trehalose (e.g., 5, 6, 7, 8, 9% w / v or higher), can maintain the purity of the antibody or its antigen-binding fragment in the formulation at 97% after incubation at 40 °C for 4 weeks.

[0118] In some embodiments, the composition further comprises a buffer (e.g., 5 - 20 mM). In some embodiments, the buffer is a citrate buffer (e.g., sodium citrate). In some embodiments, the composition comprises from about 5 mM to about 20 mM of a citrate buffer. In some embodiments, the composition comprises less than 20 mM of a citrate buffer. In some embodiments, the composition comprises less than 15 mM of a citrate buffer. In some embodiments, the composition comprises about 10 mM of a citrate buffer.

[0119] In some embodiments, the composition is buffered to a pH of about 4.0 to 6.0. In some embodiments, the composition is buffered to a pH of about 4.5 to 6.0. In some embodiments, the composition is buffered to a pH of about 5.0 to 6.0. In some embodiments, the composition is buffered to a pH of about 4.5 to 5.5. In some embodiments, the composition is buffered to a pH of about 5.0 to 5.5. In some embodiments, the composition is buffered to a pH of about 5.2.

[0120] In some embodiments, the composition further comprises a surfactant (e.g., 0.005 - 0.100% (w / v)). In some embodiments, the surfactant comprises a nonionic surfactant (e.g., 0.005 - 0.100% (w / v)). In some embodiments, the surfactant comprises polysorbate 80. In some embodiments, the composition comprises from about 0.005% (w / v) to about 0.100% polysorbate 80. In some embodiments, the composition comprises from about 0.010% (w / v) to about 0.080% polysorbate 80. In some embodiments, the composition comprises from about 0.010% (w / v) to about 0.050% polysorbate 80. In some embodiments, the composition comprises from about 0.010% (w / v) to about 0.030% polysorbate 80. In some embodiments, the composition comprises from about 0.015% (w / v) to about 0.025% polysorbate 80. In some embodiments, the composition comprises about 0.020% (w / v) of polysorbate 80.

[0121] In some embodiments, the composition further comprises a chelating agent (e.g., 0.01 - 0.10 mM). In some embodiments, the chelating agent comprises EDTA. In some embodiments, the composition comprises from about 0.001 mM to about 0.350 mM EDTA. In some embodiments, the composition comprises from about 0.010 mM to about 0.100 mM EDTA. In some embodiments, the composition comprises from about 0.020 mM to about 0.080 mM EDTA. In some embodiments, the composition comprises from about 0.040 mM to about 0.60 mM EDTA. In some embodiments, the composition comprises about 0.050 mM EDTA.

[0122] In some embodiments, the composition comprises:

[0123] An anti - ROR1 or an ROR1 - binding antibody fragment thereof at a concentration of about 40 milligrams per milliliter;

[0124] About 10 mM sodium citrate;

[0125] About 0.05 mM EDTA;

[0126] About 0.02% (w / v) of polysorbate 80; and

[0127] About 7.5% (w / v) trehalose (e.g., trehalose - 2H 2 O);

[0128] The pH of the composition is about 5.2.

[0129] In some embodiments, the composition is for intravenous injection. In some embodiments, the composition is administered by intravenous injection. Other injection routes that can be considered include, for example, subcutaneous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, or intracerebral injection, etc.

[0130] Method

[0131] The compositions described herein can be useful and / or advantageous in methods for treating cancer. Methods for treating cancer or tumors are provided herein, which include:

[0132] administering to an individual in need a composition comprising an anti-ROR1 antibody or an ROR1-binding fragment thereof as described herein.

[0133] For example, methods for treating cancer or tumors are provided, the methods comprising:

[0134] administering a composition comprising an anti-ROR1 antibody or an ROR1-binding antibody fragment thereof; and about 5% (w / v) or more trehalose; wherein the anti-ROR1 antibody or the ROR1-binding antibody fragment comprises:

[0135] (a) heavy chain complementarity determining region 1 (H-CDR1) which comprises the amino acid sequence shown in SEQ ID NO: 1;

[0136] (b) heavy chain complementarity determining region 2 (H-CDR2) which comprises the amino acid sequence shown in SEQ ID NO: 2;

[0137] (c) heavy chain complementarity determining region 3 (H-CDR3) which comprises the amino acid sequence shown in SEQ ID NO: 3;

[0138] (d) light chain complementarity determining region 1 (L-CDR1) which comprises the amino acid sequence shown in SEQ ID NO: 4;

[0139] (e) light chain complementarity determining region 2 (L-CDR2) which comprises the amino acid sequence shown in SEQ ID NO: 5; and / or

[0140] (f) light chain complementarity determining region 3 (L-CDR3) which comprises the amino acid sequence shown in SEQ ID NO: 6.

[0141] In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the leukemia or lymphoma is B-cell leukemia or lymphoma. In some embodiments, the leukemia or lymphoma is chronic lymphocytic leukemia (CLL). In some embodiments, the leukemia or lymphoma is mantle cell lymphoma (MCL).

[0142] In some embodiments, the cancer is a solid tissue cancer and / or comprises a solid tumor. In some embodiments, the solid tissue cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, head and neck cancer, kidney cancer, colon cancer, or gastric cancer.

[0143] Definitions

[0144] Antibodies are used in the broadest sense and generally encompass and / or refer to various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity. In some embodiments, one or more antibodies include intact antibodies and their functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab) 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single-chain antibody fragments, including single-chain variable fragments (sFv or scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. In some embodiments, one or more antibodies include genetically engineered and / or other modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetra-bodies, tandem bis-scFv, tandem tris-scFv. In some embodiments, one or more antibodies comprise their functional antibody fragments. In some embodiments, one or more antibodies comprise intact or full-length antibodies, including any class or subclass of antibodies, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region. In some embodiments, one or more antibodies include but are not limited to full-length and native antibodies, as well as fragments and portions that retain their binding specificity, such as having any number of any specific binding moieties, including immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant (antigen-binding) fragments or their specific binding moieties, including but not limited to Fab, F(ab) 2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally a single type in a substantially homogeneous antibody composition; thus, any individual antibody contained in a monoclonal antibody composition is the same, except for natural mutations that may be present in small amounts in the composition. Monoclonal antibodies can contain the human IgG1 constant region or the human IgG4 constant region.

[0145] Natural antibodies generally contain and / or refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies can be monomeric or polymeric glycoproteins composed of two identical light chains and two identical heavy chains, which are linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by the constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be divided into two types, called kappa (κ) type and lambda (λ) type.

[0146] Full-length antibodies, intact antibodies, and whole antibodies are interchangeable and generally include and / or refer to antibodies having a structure substantially similar to that of natural antibodies or having heavy chains that contain an Fc region as defined herein.

[0147] Human consensus frameworks generally contain and / or refer to frameworks that represent the amino acid residues most frequently occurring in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, this sequence subgroup is the subgroup described in "Sequences of Proteins of Immunological Interest", 5th Edition, NIH Publication 91-3242, Bethesda, Maryland (1991), Volumes 1-3, by Kabat et al. In some embodiments, for VL, this subgroup is the kappa I subgroup as described by Kabat et al. in the above-mentioned literature. In some embodiments, for VH, this subgroup is the III subgroup as described by Kabat et al. in the above-mentioned literature. Humanized antibodies contain and refer to chimeric antibodies that contain amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs). In some embodiments, a humanized antibody will include at least one, and typically two, variable regions, wherein all or substantially all HVRs (e.g., CDRs) correspond to the corresponding portions of a non-human antibody, and all or substantially all FRs correspond to the corresponding portions of a human antibody. A humanized antibody optionally contains at least a portion of an antibody constant region derived from a human antibody. An "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

[0148] Complementary determining regions or CDRs, synonymous with hypervariable regions or HVRs, generally comprise and / or refer to regions of an antibody that are highly variable in sequence and / or form structurally defined loops (hypervariable loops) and / or contain residues that contact an antigen (the antigen). In some embodiments, the complementary determining regions or CDRs generally comprise and refer to discontinuous amino acid sequences within the variable regions of an antibody that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3).

[0149] The framework region or FR generally comprises and / or refers to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. In some embodiments, the framework region is defined by the non-CDR sequences of the variable region sequences.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of known numbering schemes, including those described in Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); the scheme described in Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); the "Antibody-antigen interactions: Contact analysis and binding site topography" scheme in MacCallum et al., in J. Mol. Biol. 262:732-745 (1996) ("Contact" numbering scheme); the scheme described in Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, January 2003; 27(1):55-77 ("IMGT" numbering scheme); the scheme described in Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, June 8, 2001; 309(3):657-70 ("Aho" numbering scheme); the scheme described in Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB", Protein Eng. December 2000; 13(12):819-24 ("AbM" numbering scheme).In some embodiments, the CDRs of the antibodies described herein can be defined by methods selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof. In some embodiments, the CDRs and FRs are defined by and / or according to the Kabat numbering scheme. In some embodiments, the CDRs and FRs are defined by and / or according to the Chothia numbering scheme. In some embodiments, the CDRs and FRs are defined by and / or according to the IMGT numbering scheme. In some embodiments, the CDRs and FRs are defined by and / or according to the EU numbering scheme.

[0150] In some cases, the boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions accommodated by inserting letters (e.g., "30a"), and deletions can occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0151] The Fc region generally comprises and / or refers to the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, Maryland (1991).

[0152] A human antibody generally comprises and / or refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or an antibody from a non-human source derived using a human antibody library or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0153] The acceptor human framework region generally comprises and / or refers to a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. The acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the identical amino acid sequence thereof, or it may comprise amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0154] The variable region or variable domain generally comprises and / or refers to the domain of an antibody heavy chain or light chain that participates in antibody-antigen binding. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., “Kuby Immunology”, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. In addition, the VH or VL domain from an antibody that binds a particular antigen can be used separately to screen a library of complementary VL or VH domains to isolate antibodies that bind that antigen (see, e.g., Portolano et al., J. Immunol. 150:880--887, 1993; Clarkson et al., Nature 352:624-628, 1991).

[0155] Affinity generally comprises and / or refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, binding affinity as used herein generally comprises and refers to intrinsic binding affinity, which reflects the 1:1 interaction between a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments of measuring binding affinity are described throughout the specification.

[0156] An affinity matured antibody generally comprises and / or refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parental antibody that does not have such alterations, such alterations resulting in an increase in the affinity of the antibody for the antigen.

[0157] Binding and its determination can be readily determined by methods known in the art (e.g., enzyme-linked immunosorbent assay, surface plasmon resonance, biolayer interferometry, isothermal calorimetry, etc.). In some embodiments, the binding is determined by ELISA assay. In some embodiments, binding with a KD of less than, for example, 10 -5 M (10 μM) is measured by surface plasmon resonance, biolayer interferometry, or isothermal calorimetry. In some embodiments, binding with a KD of less than, for example, 10 -6 M (1 μM) is measured by surface plasmon resonance, biolayer interferometry, or isothermal calorimetry. In some embodiments, binding with a KD of less than, for example, 10 -7 M (100 nM) is measured by surface plasmon resonance, biolayer interferometry, or isothermal calorimetry.

[0158] Chimeric antibodies generally include and / or refer to antibodies in which a portion of the heavy chain and / or light chain is from a particular source or species, while the remaining portion of the heavy chain and / or light chain is from a different source or species. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA 1 and IgA 2 . The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated a, d, e, g, and m, respectively.

[0159] Monoclonal antibodies generally include and / or refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies making up the population are identical and / or bind the same epitope, although variants may exist, such as those containing naturally occurring mutations or those generated during the preparation of the monoclonal antibody, and such variants are generally present in small amounts in the composition. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. In some embodiments, the modifier monoclonal indicates the characteristic that the antibody is obtained from a substantially homogeneous antibody population and should not be construed as requiring any particular method for producing the antibody. For example, monoclonal antibodies used in a composition according to the present disclosure can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0160] Isolated antibodies generally include and / or refer to antibodies that have been separated from the components of their natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0161] In some embodiments, the antibody comprises one or more naturally occurring amino acids. In some embodiments, the antibody consists of naturally occurring amino acids. As used herein, naturally occurring amino acids include and / or refer to amino acids that are generally found in nature and have not been artificially manipulated. In certain instances, naturally occurring further includes and / or refers to the 20 conventional amino acids: alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0162] In some embodiments, the antibody comprises a variant sequence of the antibody. In certain cases, amino acid substitutions can be made in any of the antibody sequences described herein without necessarily reducing or eliminating its activity (e.g., as measured by the binding or functional assays described herein). Thus, in some embodiments, the variant sequence comprises one or more amino acid substitutions. In some embodiments, the variant sequence comprises one or more substitutions in one or more complementarity-determining regions (CDRs). In some embodiments, the variant sequence comprises one amino acid substitution. In some embodiments, the variant sequence comprises two amino acid substitutions. In some embodiments, the variant sequence comprises three amino acid substitutions. In certain cases, the substitutions include conservative substitutions (e.g., substitution with an amino acid having comparable chemical properties). In certain cases, a nonpolar amino acid can be replaced and substituted with another nonpolar amino acid, where the nonpolar amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. In certain cases, a neutrally charged polar amino acid can be replaced with another neutrally charged polar amino acid, where the neutrally charged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In certain cases, a positively charged amino acid can be replaced with another positively charged amino acid, where the positively charged amino acids include arginine, lysine, and histidine. In certain cases, a negatively charged amino acid can be replaced and substituted with another negatively charged amino acid, where the negatively charged amino acids include aspartic acid and glutamic acid. Examples of amino acid substitutions also include replacing its corresponding D-amino acid with an L-amino acid, and replacing a homosteroid or other unnatural amino acid with cysteine.

[0163] In some embodiments, the antibody comprises one or more non-natural amino acids. In some embodiments, the antibody consists of non-natural amino acids. As used herein, non-natural amino acids and / or non-natural amino acids include and / or refer to amino acid structures that cannot be biosynthetically produced using unmodified or modified genes from any organism in any organism. For example, these include, but are not limited to, modified amino acids and / or amino acid analogs that are not one of the 20 naturally occurring amino acids (e.g., non-natural side chain variant sequence amino acids), D-amino acids, homoisoamino acids, β-homoisoamino acids, N-methyl amino acids, or α-methyl amino acids. Further by way of example, non-natural amino acids also include 4-benzoyl-L-phenylalanine (Bpa), anthranilic acid (Abz), aminobutyric acid (Abu), aminohexanoic acid (Ahx), amino isobutyric acid (Aib), citrulline (Cit), diaminobutyric acid (Dab), diaminopropionic acid (Dap), diaminopropionic acid (Dap), γ-carboxyglutamic acid (Gia), homoisoleucine (Hala), homoarginine (Harg), homoasparagine (Hasn), homoserine (Hasp), homocysteine (Heys), homoglutamic acid (Hglu), homoglutamine (Hgln), homoisoleucine (Hile), homoleucine (Hleu), homomethionine (Hmet), homophenylalanine (Hphe), homoserine (Hser), homotyrosine (Htyr), homovaline (Hval), hydroxyproline (Hyp), isonipecotic acid (Inp), N-naphthylalanine (Nal), norvaline (Nip), norleucine (Nle), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), phenylglycine (Phg), pyroglutamic acid (Pyr), sarcosine (Sar), tert-butylglycine (Tie), and tetrahydroisoquinoline-3-carboxylic acid (Tic). Such non-natural amino acid residues can be introduced by substituting naturally occurring amino acids, and / or by inserting non-natural amino acids into a naturally occurring antibody sequence. Non-natural amino acid residues can also be added to endow the apelin molecule with desired functionality, such as the ability to conjugate functional molecules (such as PEG).

[0164] ROR1 generally refers to and includes the human ROR1 protein or the gene encoding the ROR1 protein (synonyms: tyrosine-protein kinase transmembrane receptor ROR1, EC = 2.7.10.1, neurotrophic tyrosine kinase receptor-related 1. UniPrtKB Q01973), which is a tyrosine-protein kinase receptor. The extracellular domain of ROR1 consists of amino acids 30 - 406. An anti-ROR1 antibody or antibodies generally refer to and include antibodies that specifically bind to human ROR1 (e.g., ELISA, surface plasmon resonance, biolayer interferometry, isothermal calorimetry, etc.). In some embodiments, the antibody specifically binds to the extracellular domain of ROR1. In certain embodiments, the antibody specifically binds to a fragment of the extracellular domain, namely the Ig-like C2-type domain, the cysteine-rich domain, or the three-loop domain. These fragments are mentioned in W02005100605. It is further disclosed that the antibody can specifically bind to the extracellular domain fragment of ROR1, WNISSELNKDSYLTL. This fragment is disclosed in Daneshmanesh A H et al., International Journal of Cancer, 123 (2008) 1190 - 1195.

[0165] A stable formulation refers to and / or comprises a formulation in which a protein (such as an antibody) substantially retains its physical stability and / or chemical stability and / or biological activity when stored at the intended storage temperature (such as 2–8 °C). In some embodiments, the formulation substantially retains its physical and chemical stability and biological activity upon storage. The storage period can generally be selected according to the intended shelf life of the formulation. The storage period can generally be selected according to the intended shelf life of the formulation. In addition, the formulation is generally stable upon freezing (such as to -20 °C) and after thawing of the formulation, such as after undergoing one or more freeze-thaw cycles. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery (247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., published in 1991) and Jones, A., Advanced Drug Delivery Reviews 10:29-90 (1993). Stability can be measured at selected temperatures over selected time periods. Stability can be qualitatively and / or quantitatively evaluated in a variety of different ways, including assessing aggregate formation (such as using size exclusion chromatography, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity by cation exchange chromatography or capillary zone electrophoresis; SDS-PAGE analysis to compare reduced and intact antibodies; assessing the biological activity or antigen-binding function of the antibody; and the methods described herein. Instability may involve one or more of the following: aggregation, deamidation (such as Asn deamidation), oxidation (such as Met oxidation), isomerization (such as Asp isomerization), cleavage / hydrolysis / fragmentation (such as hinge region fragmentation), succinimide formation, unpaired cysteines, etc.

[0166] A pharmaceutically acceptable carrier generally includes and / or refers to a non-toxic component to the subject other than the active ingredient in a pharmaceutical formulation. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0167] Polypeptide and protein can be used interchangeably and generally include and / or refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, such as linkers and binding peptides, can include amino acid residues, including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, a polypeptide can contain modifications relative to the native or natural sequence as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host producing the protein or due to errors in PCR amplification.

[0168] Cancers expressing ROR1 or cancer cells expressing ROR1 generally refer to and include all tumor cell growth and proliferation that express, overexpress, or aberrantly express ROR1, whether malignant or benign, including all transformed cells and tissues and all cancer cells and tissues.

[0169] Mathematical algorithms can be used to determine the percent identity or percent similarity between two sequences. Preferred non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, which was modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. Alternatively, PSI-BLAST is used to perform an iterative search that detects remote relationships between molecules. When using the BLAST, Gapped BLAST, and PSL-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1989, CABIOS. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. Other algorithms known in the art for sequence analysis include ADVANCE and ADAM as described by Torellis and Robotti in 1994, Comput. Appl. Biosci. 10:3-5; and FASTA as described by Pearson and Lipman in 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Alternatively, the CLUSTAL algorithm (e.g., as provided in the program Clustal-omega) can be used for sequence alignment as described by Higgins et al. in 1996, Methods Enzymol. 266:383-402.

[0170] As used herein, the terms individual, patient, or subject generally include and / or refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease, disorder, or condition that can be treated by the compositions and methods described herein. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In some embodiments, the individual is a human.

[0171] As used herein, treatment generally comprises and / or refers to a pharmaceutical or other intervention regimen for obtaining a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic and / or prophylactic benefits. A therapeutic benefit may refer to eradicating or ameliorating the symptoms or underlying condition being treated. Additionally, a therapeutic benefit can be achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying condition such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying condition. Prophylactic effects include delaying, preventing, or eliminating the onset of a disease or disorder, delaying or eliminating the onset of symptoms of a disease or disorder, slowing, halting, or reversing the progression of a disease or disorder, or any combination thereof. For a prophylactic benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may receive treatment even if a diagnosis of the disease has not yet been made. One of ordinary skill in the art will recognize that not all individuals in a group that may receive treatment will respond to the treatment or respond equally. Such individuals are considered to have been treated.

[0172] As used herein, the terms "modulate" and "regulate" may be used interchangeably. In some embodiments, modulate includes reducing and / or decreasing and / or inhibiting. In some embodiments, modulate includes increasing and / or inducing and / or promoting and / or activating.

[0173] As used herein, the terms "comprising" (and any form of comprising, such as "comprises" and "comprising"), "having" (and any form of having, such as "has" and "having"), "including" (and any form of including, such as "includes" and "including"), or "containing" (and any form of containing, such as "contains" and "containing") are inclusive or open-ended and do not exclude other unrecited elements or method steps. As also used herein, in any instance or embodiment described herein, including may be replaced with "consisting essentially of" and / or "consisting of". In any instance or embodiment described herein, "comprising" may be replaced with "consisting essentially of" and / or "consisting of".

[0174] As used herein, in the context of a given value or range, the term "about" includes and / or refers to a value or range within 20%, within 10%, and / or within 5% of the given value or range.

[0175] As used herein, "and / or" as used herein shall be taken as specifically disclosing each of the two specified features or components, with or without the other. For example, "A and / or B" shall be taken as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if each were set forth separately herein.

[0176] Examples

[0177] Example 1 - Formulation

[0178] The antibody drug substance (ADS) from 2000 liters (L) of GMP is produced from the new master cell bank (MCB). The manufacturing process and formulation of the ADS are shown in Table 1, which includes the new formulation 1.

[0179] Table 1

[0180]

[0181] A comparability study was conducted on these two samples, focusing on the following attributes: primary structure, post-translational modifications, secondary / tertiary structure, disulfide bonds, free thiol content, binding affinity, charge heterogeneity analyzed by iCIEF, and size heterogeneity analyzed by CE-SDS and SEC-MALS. In addition, a thermal stress study was conducted at 40 °C to support the comparability assessment of these two batches of drug substances. SE-HPLC, iCIEF, non-reducing CE-SDS, reducing CE-SDS, and binding ELISA were used for analysis to compare their degradation trends.

[0182] Example 2 - Complete Quality Analysis

[0183] Complete quality analysis provides important information on sequence integrity and major post-translational modifications. To perform molecular weight analysis of the complete protein by liquid chromatography - mass spectrometry, the sample was diluted with water to 0.4 mg / mL and then subjected to UPLC separation and Xevo G2-XS Q-TOF mass spectrometry detection. For the deconvoluted complete mass of the two formulation samples, the major species observed corresponded to the complete ADS, which was modified with pyroglutamic acid (pyro-Glu, Q converted to pE) from glutamine (Gln), glycosylated (G0F, G1F), and the C-terminal lysine (Lys) was absent on each heavy chain.

[0184] The masses of other expected glycoforms were also confirmed. All identified and assigned species showed measured masses within 100.0 ppm of the theoretical values, indicating that the complete mass was consistent with the theoretical mass. The half-antibody (LC / HC) peak of ADS formulation 2 was observed (Table 2, sample ID2, italic), but the half-antibody (LC / HC) peak with a 7.5-fold increase in trehalose was not present in the ADS formulation sample I (Table 2 - sample ID1).

[0185] Table 2

[0186]

[0187] The deglycosylated intact mass was obtained through the following process: diluting the sample with water, then performing enzymatic PNGase F digestion to remove N-glycans, followed by UHPLC separation and Xevo G2-XS Q-TOF MS detection. The deconvoluted deglycosylated mass spectrometry data of the samples are shown in Table 3. The results indicate that the measured deglycosylated intact molecular weight values of the DS samples are comparable and highly consistent with the theoretical deglycosylated molecules. However, the peak of the half-antibody (LC / HC) was observed again in ADS formulation 2 (Table 3 - sample ID2, italic), and the LC / HC peak was absent in ADS formulation sample 1 (Table 3 - sample ID1) with increased (7.5-fold) trehalose.

[0188] Table 3

[0189]

[0190] Example 3 - Analysis of Molecular Species Distribution by SEC-MALS

[0191] Size-exclusion chromatography - multi-angle light scattering (SEC-MALS) determines the absolute molar mass and average size of molecules in solution by determining the light scattering properties of substances eluted from a size-based chromatographic system. A multi-angle light scattering detector connected to a high-performance liquid chromatography - size-exclusion chromatography system was used to determine the molecular mass of ADS samples (formulation 1 and formulation 2).

[0192] The static multi-angle light scattering (MALS) instrument characterizes the absolute molecular weight of proteins based on the principle of static light scattering. For proteins larger than 10 nm, the laser scattering intensity is proportional to the molecular weight and protein concentration, and the molecular weight is calculated according to the Zimm equation.

[0193] The SEC-MALS chromatogram data of the ADS samples are shown in Table 4 and Figure 1 as follows. Molecular weight species (peaks 1 and 2) are present in both samples. The molar masses of peaks 1 and 2 in both samples are comparable. The results of SEC-MALS indicate that the molecular weight of the main aggregate (peak 1) is in the range of 294 kDa to 308 kDa, probably a dimer formed by 2 antibody protein molecules. ADS formulation sample 1 with increased (7.5-fold) trehalose exhibits less high molecular weight substance / aggregate (i.e., lower peak 1). In addition, light scattering and UV peak analysis show that ADS formulation 1 shows a reduced aggregation peak (peak 1), where the aggregation peak 1 is reduced by more than 2-fold in both peak measurements. ADS formulation 1 reduces the aggregates by more than half.

[0194] Table 4

[0195]

[0196] Example 4 - Determination of Thermal Stability by Differential Scanning Calorimetry

[0197] The thermal stability of the ADS samples (Formulations 1 and 2) was evaluated using differential scanning calorimetry (DSC), which determines the thermal transition temperature (Tm) of the protein. A higher Tm value indicates increased thermal stability, and the temperature at which the protein begins to unfold is the onset melting temperature (Tm onset). Tm and Tm onset are sensitive to changes in protein structure and stability and can be affected by differences in the formulation matrix.

[0198] DSC analysis of the ADS formulation samples 1 and 2 was performed using a Malvern / MicroCal PEAQ DSC system. The samples were heated from 10 °C to 95 °C at a heating rate of 90 °C / h. The differential scanning calorimetry scan results are shown in Table 5. The ADS formulation sample 1 exhibited a higher Tm value, which can be attributed to the formulation.

[0199] Table 5

[0200]

[0201] Example 5 - Thermal Stress Testing

[0202] The ADS samples were tested in a thermal stress study. The ADS samples were thawed at room temperature and then transferred to 1.5 mL microcentrifuge tubes in a biosafety cabinet. The fill volume was 1 mL per vial. Four vials of each ADS sample were prepared, and one vial of each DS sample was taken as the initial control. The remaining vials were incubated in a stability chamber at 40 °C. The duration, test points, and test items of the thermal stress study are listed in Table 6. At the end of each time point, the samples in each vial were aliquoted at 0.1 mL / vial, placed in a biosafety cabinet, and stored at -70 ± 10 °C.

[0203] Size exclusion - high performance liquid chromatography (SE - HPLC) was used to monitor the monomer, HMW, and LMW of the ADS samples. A 40 μg sample was injected into an Agilent 1260 system for SE - HPLC. The HPLC system was equipped with a TSKGel G3000SWXL column (5 μm, 7.8 x 300 mm) and a UV detector set at 280 nm. Isocratic elution was applied at a flow rate of 0.5 mL / min for 35 min.

[0204] The SE-HPLC results are summarized in Table 6. The results of SEC-HPLC showed that compared with ADS formulation sample 2, ADS formulation sample 1 with increased (7.5-fold) trehalose and decreased sodium citrate buffer concentration (from 50 mM to 10 mM) showed less high molecular weight substances / aggregates (i.e., HMW peak). The average difference was greater than 2-fold, and when considering all time points, ADS formulation 1 reduced the average aggregation by more than half. In addition, the percentage change in the HMW peak of ADS formulation 1 was 0.6%, while that of ADS formulation 2 was 1.6% (2.6 times higher). The same data are shown in Figure 2 in graphical form. In Figure 2 it can be observed that compared with ADS formulation sample 1, ADS formulation sample 2 showed a higher rate of aggregate formation during the heat stress study and showed a higher derivative dA / dt of the measured rate of aggregate formation over time

[0205] In addition, the SE-HPLC method was also used to measure the degradation rate of the drug product that occurred during the heat stress study. The results of SEC-HPLC showed that during the study, the drug purity of ADS formulation sample 1 decreased from 99% to 97.3%, while the drug purity of ADS formulation sample 2 decreased from 98.1% to 95.1% during the study. The average difference in the drug purity decline rate between ADS formulation sample 2 and ADS formulation sample 1 was about 1.6-fold, and the drug degradation rate of ADS formulation sample 1 decreased by about 40%. The same data are shown in Figure 2 in graphical form.

[0206] Table 6

[0207]

[0208] Example 6 - Free Sulfhydryl Analysis

[0209] The levels of free sulfhydryl groups in the ADS samples were determined because free sulfhydryl groups may indicate cases of disulfide bond mismatch and incorrect protein folding. A provided thiol analysis kit was used to determine the free sulfhydryl concentration. This analysis involves the reaction of a fluorescent reagent with the free sulfhydryl groups in the sample, generating a fluorescent signal related to the amount of free sulfhydryl groups.

[0210] In this experiment, the ADS samples were diluted 2-fold, 4-fold, and 8-fold respectively. The theoretical molecular weight of the ADS protein is 147723.4 Da. The free SH concentration in the samples was calculated. As shown in Table 7, the free SH contents in the ADS samples were 0.54% and 5.47% respectively. The level of free sulfhydryl groups in ADS formulation sample 1 with increased (7.5-fold) trehalose was about 10 times lower than the level observed in ADS formulation sample 2, indicating more accurate protein folding in ADS formulation sample 1.

[0211] Table 7

[0212] ID Free thiol content (μM / μM protein) 1 0.54％ 2 5.47％

[0213] While the preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art can make various changes, variations and substitutions without departing from the present invention. It should be understood that various alternative forms of the embodiments of the present invention described herein can be used for the present invention. The appended claims define the scope of the present invention, and the methods and structures within the scope of these claims, as well as their equivalents, are included in the present invention.

[0214] Sequence

[0215]

[0216]

Claims

1. A composition comprising: a ROR1 antibody or antigen-binding fragment thereof at a concentration of about 20 to about 80 mg / ml; Buffer; Chelating agents; surfactant; and 5.0% (w / v) or higher concentration of trehalose dihydrate; in, The pH of the composition is 6.0 or less.

2. The composition according to claim 1, in, The composition reduces the formation of aggregates.

3. The composition according to claim 2, in, The aggregates comprise a precipitate and the composition reduces the formation of the precipitate.

4. The composition according to claim 1, in, The composition contains less than 2% antibody aggregates.

5. The composition according to claim 1, in, The composition contained less than 2% antibody aggregates after incubation at 40°C for 4 weeks.

6. The composition according to claim 1, in, The composition maintains the purity of the antibody or antigen-binding fragment thereof.

7. The composition according to claim 1, in, The composition maintains at least 97% purity of the antibody or antigen-binding fragment thereof.

8. The composition according to claim 1, in, The composition retains at least 97% purity of the antibody or antigen-binding fragment thereof after incubation at 40°C for 4 weeks.

9. The composition according to claim 1, in, The antibody is Zelotuzumab.

10. The composition according to any one of claims 1 to 9, in, The buffer includes acetate, citrate, histidine, arginine, succinate or phosphate.

11. The composition of claim 10, wherein the buffer comprises sodium citrate.

12. A composition according to any one of claims 1 to 11, in, The buffer is present in the composition at a concentration of about 5 mM to about 100 mM.

13. A composition according to any one of claims 1 to 11, in, The buffer is present in the composition at a concentration of about 5 mM to about 50 mM.

14. A composition according to any one of claims 1 to 11, in, The concentration of the buffer in the composition is about 5 mM to about 20 mM.

15. A composition according to any one of claims 1 to 11, in, The concentration of the buffer in the composition is about 10 mM.

16. A composition according to any one of claims 1 to 11, in, The buffer may be present in the composition at a concentration of up to 10 mM.

17. The composition of claim 16, in, The buffer is sodium citrate.

18. The composition according to any one of claims 1 to 15, in, The chelating agent includes ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA).

19. The composition of claim 18, in, The chelating agent comprises EDTA.

20. The composition according to any one of claims 1 to 19, in, The chelating agent is present in the composition at a concentration of about 0.01 mM to about 0.2 mM.

21. The composition according to any one of claims 1 to 19, in, The chelating agent is present in the composition at a concentration of about 0.01 mM to about 0.1 mM.

22. The composition of any one of claims 1 to 19, in, The chelating agent is present in the composition at a concentration of about 0.02 mM to about 0.2 mM.

23. The composition of any one of claims 1 to 19, in, The concentration of the chelating agent in the composition is about 0.05 mM.

24. The composition of any one of claims 1 to 23, in, The surfactant includes polysorbate 80, polysorbate 20 or polyethylene-polypropylene copolymer.

25. The composition of any one of claims 1 to 23, in, The surfactant comprises polysorbate 80.

26. The composition of any one of claims 1 to 25, in, The surfactant is present in the composition at a concentration of about 0.005% (w / v) to about 0.1% (w / v).

27. The composition of any one of claims 1 to 25, in, The surfactant is present in the composition at a concentration of about 0.01% (w / v) to about 0.05% (w / v).

28. A composition as claimed in any one of claims 1 to 25, in, The surfactant is present in the composition at a concentration of about 0.01% (w / v) to about 0.03% (w / v).

29. The composition of any one of claims 1 to 25, in, The concentration of the surfactant in the composition is about 0.02% (w / v).

30. The composition of any one of claims 1 to 29, in, The concentration of trehalose dihydrate in the composition is about 5.0% (w / v) to about 10.0% (w / v).

31. A composition as claimed in any one of claims 1 to 29, in, The concentration of trehalose dihydrate in the composition is about 6.0% (w / v) to about 9.0% (w / v).

32. A composition as claimed in any one of claims 1 to 29, in, The concentration of trehalose dihydrate in the composition is about 7.0% (w / v) to about 8.0% (w / v).

33. A composition according to any one of claims 1 to 29, in, The concentration of trehalose dihydrate in the composition is about 7.5% (w / v).

34. A composition as claimed in any one of claims 1 to 33, in, The pH of the composition is from about 4.0 to about 6.

0.

35. A composition as claimed in any one of claims 1 to 33, in, The pH of the composition is from about 4.5 to about 6.

0.

36. A composition as described in any one of claims 1 to 33, in, The pH of the composition is from about 4.0 to about 5.

5.

37. A composition as described in any one of claims 1 to 33, in, The pH of the composition is from about 4.5 to about 5.

5.

38. A composition as described in any one of claims 1 to 33, in, The pH of the composition is from about 5.0 to about 5.

5.

39. A composition as described in any one of claims 1 to 33, in, The pH of the composition was about 5.

2.

40. The composition of any one of claims 1 to 39, in, The composition is a liquid.

41. A method of treating cancer comprising administering to an individual in need thereof a composition as claimed in any one of claims 1 to 40.

42. The method of claim 41, in, The cancer is leukemia or lymphoma.

43. The method of claim 42, in, The leukemia or lymphoma is a B-cell leukemia or lymphoma.

44. The method of claim 42, in, The leukemia or lymphoma is chronic lymphocytic leukemia (CLL).

45. The method of claim 42, in, The leukemia or lymphoma is mantle cell lymphoma (MCL).

46. ​​The method of claim 41, in, The cancer is a solid tissue cancer.

47. The method of claim 46, in, The solid tissue cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, head and neck cancer, kidney cancer, colon cancer or gastric cancer.

48. A composition comprising: A ROR1 antibody or antigen-binding fragment thereof at a concentration of about 40 mg / mL; About 10 mM sodium citrate; About 0.05 mM EDTA; about 0.02% (w / v) polysorbate 80; and About 7.5% (w / v) trehalose dihydrate; The pH of the composition is about 5.

2.

49. The composition of claim 48, in, The composition reduces the formation of aggregates.

50. The composition of claim 49, in, The aggregates comprise a precipitate and the composition reduces the formation of the precipitate.

51. The composition of claim 48, in, The composition contains less than 2% antibody aggregates.

52. The composition of claim 48, in, The composition contained less than 2% antibody aggregates after incubation at 40°C for 4 weeks.

53. The composition of claim 48, in, The composition maintains the purity of the antibody or antigen-binding fragment thereof.

54. The composition of claim 48, in, The composition maintains at least 97% purity of the antibody or antigen-binding fragment thereof.

55. The composition of claim 48, in, The composition retains at least 97% purity of the antibody or antigen-binding fragment thereof after incubation at 40°C for 4 weeks.

56. The composition of claim 48, in, The antibody is Zelotuzumab.

57. A composition comprising: an anti-ROR1 antibody or a ROR1 -binding antibody fragment thereof at a concentration of about 20 to about 80 milligrams / milliliter (mg / mL); and Trehalose at a concentration of about 5% (w / v) or more; in, The anti-ROR1 antibody or ROR1 binding antibody fragment thereof comprises: (a) a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence shown in SEQ ID NO: 1; (b) a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid sequence shown in SEQ ID NO: 2; (c) a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid sequence shown in SEQ ID NO: 3; (d) a light chain complementary determining region 1 (L-CDR1) comprising the amino acid sequence shown in SEQ ID NO:4; (e) a light chain complementary determining region 2 (L-CDR2) comprising the amino acid sequence shown in SEQ ID NO: 5; and / or (f) a light chain complementary determining region 3 (L-CDR3) comprising the amino acid sequence shown in SEQ ID NO: 6; wherein the anti-ROR1 antibody or the ROR1 binding antibody fragment thereof binds to ROR1.

58. The composition of claim 57, in, The composition reduces the formation of aggregates.

59. The composition of claim 58, in, The aggregates comprise a precipitate and the composition reduces the formation of the precipitate.

60. The composition of claim 57, in, The composition contains less than 2% antibody aggregates.

61. The composition of claim 57, in, The composition contained less than 2% antibody aggregates after incubation at 40°C for 4 weeks.

62. The composition of claim 57, in, The composition maintains the purity of the antibody or antigen-binding fragment thereof.

63. The composition of claim 57, in, The composition maintains at least 97% purity of the antibody or antigen-binding fragment thereof.

64. The composition of claim 57, in, The composition retains at least 97% purity of the antibody or antigen-binding fragment thereof after incubation at 40°C for 4 weeks.

65. The composition of claim 57, in, The anti-ROR1 antibody or ROR1 binding antibody fragment thereof comprises: A heavy chain variable domain having 90% or greater sequence identity to SEQ ID NO:7; and A light chain variable domain having 90% or greater sequence identity to SEQ ID NO:

8.

66. The composition of claim 57, in, The anti-ROR1 antibody or ROR1 binding antibody fragment thereof comprises: A heavy chain variable domain comprising SEQ ID NO: 7; and A light chain variable domain comprising SEQ ID NO:

8.

67. The composition of claim 57, in, The anti-ROR1 antibody or ROR1 binding antibody fragment thereof comprises: A heavy chain constant domain having 90% or greater sequence identity to SEQ ID NO:

11.

68. The composition of claim 57, in, The anti-ROR1 antibody or ROR1 binding antibody fragment thereof comprises: A heavy chain domain having 90% or greater sequence identity to SEQ ID NO:9; and A light chain domain having 90% or greater sequence identity to SEQ ID NO:

10.

69. The composition of claim 57, in, The anti-ROR1 antibody or ROR1 binding antibody fragment thereof comprises: A heavy chain domain comprising SEQ ID NO:9; and A light chain domain comprising SEQ ID NO:

10.

70. A composition according to any one of claims 57 to 69, in, The anti-ROR1 antibody is zelotuzumab.

71. A composition as described in any one of claims 57 to 70, in, The concentration of the anti-ROR1 antibody or ROR1 binding antibody fragment thereof is between about 30 mg / mL and about 50 mg / mL.

72. The composition of claim 71, in, The concentration of the anti-ROR1 antibody or ROR1 binding antibody fragment thereof is about 40 mg / mL.

73. A composition as described in any one of claims 57 to 69, in, The composition comprises about 7% (w / v) or higher concentration of trehalose.

74. The composition of claim 52, in, The composition comprises about 7.5% (w / v) trehalose.

75. A composition as described in any one of claims 57 to 69, in, The composition also comprises a citrate buffer.

76. The composition of claim 69, in, The composition comprises less than about 20 mM citrate buffer.

77. The composition of claim 69, in, The composition comprises less than about 15 mM citrate buffer.

78. The composition of claim 69, in, The composition comprises about 5 mM to about 20 mM citrate buffer.

79. The composition of claim 69, in, The composition comprises about 10 mM citrate buffer.

80. The composition of claim 69, in, The composition comprises up to 10 mM citrate buffer.

81. The composition of claim 80, in, The citrate buffer is sodium citrate.

82. A composition as described in any one of claims 57 to 81, in, The composition is buffered to a pH of about 4.5 to about 5.

5.

83. A composition as described in any one of claims 57 to 82, in, The composition also comprises a nonionic surfactant.

84. The composition of claim 83, in, The nonionic surfactant is polysorbate 80.

85. A composition as claimed in claim 84, in, The composition comprises polysorbate 80 at about 0.010% (w / v) to about 0.050% (w / v).

86. A composition as described in any one of claims 57 to 85, in, The composition also comprises a chelating agent.

87. The composition of claim 86, in, The chelating agent comprises EDTA.

88. The composition of claim 86, in, The composition comprises: an anti-ROR1 antibody or a ROR1 -binding antibody fragment thereof at a concentration of about 40 mg / mL; About 7.5% (w / v) trehalose dihydrate; About 10 mM citrate buffer; about 0.02% (w / v) polysorbate 80; and About 0.05mM EDTA.

89. The composition of claim 88, in, The citrate buffer is sodium citrate.

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