Formulations comprising anti-CD 47/PD-L1 bispecific antibodies and methods of making and using thereofTechnical FieldThe present invention relates to the field of antibody formulations. More particularly, the present invention relates to pharmaceutical formulations, especially stable high concentration antibody liquid formulations, comprising recombinant anti-differentiation antigen cluster 47(CD47) and anti-programmed death ligand 1(PD-L1) bispecific antibodies (also known as anti-CD 47/PD-L1 bispecific antibodies), as well as methods for the preparation of said pharmaceutical formulations, and therapeutic and/or prophylactic uses of said pharmaceutical formulations.
BackgroundPD-L1 (also known as differentiation antigen cluster 274(CD274) or B7 homolog 1(B7-H1)) is a 40kDa type I transmembrane protein. PD-L1 binds to its receptor, PD-1, present on activated T cells, down-regulates T cell activation (Latchman et al, 2001 Nat Immunol 2: 261-8; Carter et al, 2002 Eur J Immunol 32: 634-43). PD-L1 expression has been found in many cancers, including human lung, ovarian, colon, and various myelomas, among others, and PD-L1 expression is often associated with poor prognosis of the Cancer (Iwai et al (2002) PNAS 99: 12293-7; Ohigashi et al (2005) Clin Cancer Res 11: 2947-53; Okazaki et al (2007) Intern. Immun.19: 813-24; Thompson et al (2006) Cancer Res.66: 3381-5). It has been suggested that immunosuppression may be reversed in a subset of tumor patients by inhibiting the local interaction of PD1 with PD-L1.
The anti-PD-L1 antibody Atezolizumab developed by Roche (Roche), the anti-PD-L1 antibody Avelumab developed by cooperation of Merck KGaA and Barrey (Pfizer) in Germany, and the anti-PD-L1 antibody Avelumab developed by Aslicon, and Durvalumab developed by Aslicon show therapeutic effects on a part of tumor patients. Other anti-PD-L1 antibodies include yw243.55.s70 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 in WO 2010/077634) and anti-PD-L1 antibodies disclosed in WO2007/005874, and the like.
The differentiation antigen cluster 47(CD47), also known as integrin-associated protein (IAP), is a member of the immunoglobulin superfamily. CD47 interacts with a cell surface immunoglobulin sirpa, which is expressed primarily by macrophages and dendritic cells as its ligand, to produce a cascade of reactions and thereby inhibit the uptake and phagocytosis of CD 47-expressing cells by macrophages and dendritic cells. The presence of CD47 overexpression was observed in tumors. However, CD47 is also expressed in many normal tissues, which results in nonspecific binding of antibodies targeting CD47 only to normal blood system cells, causing an antigen silencing (antigen sink) phenomenon.
The anti-CD 47/PD-L1 bispecific antibody can simultaneously target CD47 and PD-L1 on tumor cells, promotes the selective binding of the anti-CD 47/PD-L1 bispecific antibody to the tumor cells through the specific binding with the PD-L1 on the tumor cells, and avoids the binding with CD47 expressed in many normal tissues, so the anti-CD 47/PD-L1 bispecific antibody has the advantages of enhancing the anti-tumor effect and reducing side effects.
PCT application (filing date: 2018, 12 and 26) of PCT/CN2018/123886 discloses a novel antibody pattern, and constructs and expresses an anti-CD 47/PD-L1 bispecific antibody with the novel antibody pattern. The anti-CD 47/PD-L1 bispecific antibody is applied to tumor-bearing mice generated by NOD-SCID mice inoculated with Raji-PD-L1 cells, and the results show that the anti-CD 47/PD-L1 bispecific antibody has remarkably improved anti-tumor activity, can remarkably inhibit the growth of tumors and even can completely eliminate the tumors compared with the application of an anti-CD 47 monoclonal antibody and an anti-PD-L1 monoclonal antibody. Furthermore, the anti-CD 47/PD-L1 bispecific antibody also exhibits significantly reduced hemagglutination and will therefore have significantly reduced side effects in clinical treatment. There is a need in the art for anti-CD 47/PD-L1 bispecific antibody formulations that can be used to treat, prevent or delay a variety of diseases associated with the sirpa/CD 47 signaling pathway and the PD1/PD-L1 signaling pathway.
In addition to the antibody formulation being formulated in a manner that renders the antibody suitable for administration to a subject, the antibody formulation also needs to be formulated in a manner that maintains its stability during storage and subsequent use. For example, if an antibody is not properly formulated in a liquid, the antibody in the liquid solution is prone to decomposition, aggregation, or undesirable chemical modification, etc. The stability of an antibody in an antibody formulation depends on the buffers, stabilizers, surfactants, and the like used in the formulation.
Although some anti-CD 47/PD-L1 bispecific antibodies are known, there remains a need in the art for novel pharmaceutical formulations containing anti-CD 47/PD-L1 bispecific antibodies that are sufficiently stable and suitable for administration to a subject. Thus, there is a need for suitable anti-CD 47/PD-L1 bispecific antibody formulations for the treatment or prevention of disease.
Summary of The Invention
The present invention meets the above-described needs by providing a pharmaceutical formulation comprising an anti-CD 47/PD-L1 bispecific antibody protein that specifically binds to CD47 and PD-L1.
In one aspect, the invention provides a liquid antibody formulation comprising (i) an anti-CD 47/PD-L1 bispecific antibody protein; (ii) a buffering agent, (iii) a stabilizing agent, and (iv) a surfactant.
The anti-CD 47/PD-L1 bispecific antibody protein comprised in the antibody preparation of the invention is a three-chain antibody comprising a VH on a first polypeptide chain and a second polypeptide chain that specifically binds CD47a/VL pair as a first antigen binding site, and a first VHH on a third polypeptide chain that specifically binds PD-L1 as a single domain second antigen binding site and a second VHH as a single domain third antigen binding site; or a VH/VL pair on a first and second polypeptide chain that specifically binds PD-L1 as a first antigen binding site and a first VHH on a third polypeptide chain that specifically binds CD47 as a single domain second antigen binding site and a second VHH as a single domain third antigen binding site. In some embodiments, the anti-CD 47/PD-L1 bispecific antibody protein can be at least about 107M-1Preferably about 108M-1And more preferably about 109M-1Or stronger affinity constant is combined with CD47 on the surface of the tumor cell, thereby blocking the combination of CD47 and SIRP alpha on the surface of macrophage and promoting the phagocytosis of the macrophage in the tumor tissue infiltration area to the tumor cell; and at least about 107M-1Preferably about 108M-1And more preferably about 109M-1Or a stronger affinity constant, to bind to PD-L1 on the surface of tumor cells, thereby inhibiting the binding of PD-1 on T cells to PD-L1 on the surface of tumor cells, inducing T cell activation and exerting an anti-tumor effect.
In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein is a recombinant anti-CD 47/PD-L1 bispecific antibody protein disclosed in PCT application PCT/CN2018/123886 (application date: 2018, 12 months and 26 days). The entire contents of the PCT application are hereby incorporated by reference for the purposes of this application. In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein is a three chain antibody comprising as a first antigen-binding site a VH/VL pair on a first polypeptide chain and a second polypeptide chain that specifically binds CD47, and as a single domain a first VHH on a third polypeptide chain that specifically binds PD-L1 as a single domain a second antigen-binding site and a second VHH as a single domain a third antigen-binding site, wherein the VH/VL pair on the first polypeptide chain and the second polypeptide chain that specifically binds CD47 comprises as a first antigen-binding site a VH CDR1 as shown by GSIEHYYWS (SEQ ID NO:3), a VH CDR2 as shown by YIYYSGSTNYNPSLKS (SEQ ID NO:4), a VH CDR3 as shown by ARGKTGSAA (SEQ ID NO:5), a VL CDR1 as shown by RASQGISRWLA (SEQ ID NO:10), a VL CDR1 as shown by ADI-29341 derived from the anti-CD 47 antibody, VL CDR2 and QQTVSFPIT (SEQ ID NO:12) as set forth in AASSLQS (SEQ ID NO:11), or VL CDR3 as set forth in AASSLQS (SEQ ID NO:12), or a sequence having one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) with one or more of the 6 CDRs; the single domain second and third antigen-binding sites on the third polypeptide chain that specifically bind PD-L1 each comprise CDR1 shown in AYTISRNSMG (SEQ ID NO:17), CDR2 shown in IESDGST (SEQ ID NO:18) and CDR3 shown in AAPKVGLGPRTALGHLAFMTLPALNY (SEQ ID NO:19), or a sequence with one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) to one or more of the 3 CDRs,
in one embodiment, the VH/VL pair specifically binding CD47 on said first and second polypeptide chains of the anti-CD 47/PD-L1 bispecific antibody protein comprises as a first antigen-binding site a heavy chain variable region sequence/light chain variable region sequence derived from SEQ ID NO:2/9 of anti-CD 47 antibody ADI-29341, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said heavy chain variable region sequence/light chain variable region sequence, and the single domain second and third antigen-binding sites on said third polypeptide chain that specifically bind PD-L1 each comprise or are substantially identical to an amino acid sequence set forth in SEQ ID NO:15 and/or SEQ ID NO:16 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical):
in one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein comprises a first polypeptide chain as set forth in SEQ ID NO:1, a second polypeptide chain as set forth in SEQ ID NO:8, and a third polypeptide chain as set forth in SEQ ID NO:14 or SEQ ID NO:22, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any one of said sequences.
In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein is an anti-CD 47/PD-L1 bispecific antibody protein recombinantly expressed in HEK293 cells or CHO cells.
In one embodiment, the concentration of anti-CD 47/PD-L1 bispecific antibody protein in a liquid antibody formulation of the invention is about 1-200 mg/ml. In another embodiment, the concentration of anti-CD 47/PD-L1 bispecific antibody protein in a liquid antibody formulation of the invention is about 5-150 mg/mL. In other embodiments, the concentration of anti-CD 47/PD-L1 bispecific antibody protein in a liquid antibody formulation of the invention is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg/ml.
In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 1-30 mM. In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 5-25mM, e.g., about 5, 10, 15, 20, 25 mM.
In one embodiment, the buffering agent is selected from histidine, histidine hydrochloride, and combinations thereof.
In one embodiment, the concentration of the stabilizing agent in the liquid antibody formulation of the invention is about 50-500 mM. In one embodiment, the concentration of the stabilizing agent in the liquid antibody formulation of the invention is about 100-400mM, e.g., about 100, 150, 200, 250, 300, 350, 400 mM.
In one embodiment, the stabilizing agent is selected from the group consisting of sorbitol, sucrose, trehalose, arginine hydrochloride, and any combination thereof, more preferably sucrose, arginine, and/or arginine hydrochloride. In one embodiment, the liquid antibody formulation comprises arginine hydrochloride as a stabilizer, preferably arginine hydrochloride is present in an amount of about 50-250mM, preferably about 100-200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM); and/or sucrose as a stabilizer, preferably sucrose is present in an amount of about 50-250mM, preferably about 100-200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
In one embodiment, the concentration of surfactant in the liquid antibody formulation of the invention is about 0.1-1 mg/ml. In one embodiment, the concentration of surfactant in a liquid antibody formulation of the invention is about 0.2-0.8mg/ml, e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
In one embodiment, the surfactant is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate-based surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the invention is polysorbate-80.
In some embodiments, the liquid formulation further comprises a metal chelator (e.g., EDTA), for example, about 0.002-0.2 mg/ml of the metal chelator (e.g., EDTA). In one embodiment, the liquid formulation further comprises about 0.01-0.1mg/ml, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.1mg/ml, of a metal chelator (e.g., EDTA).
In one embodiment, the pH of the liquid formulation is about 6.4-7.0. In some embodiments, the pH of the liquid formulation is any of about 6.4-7.0, e.g., about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.
In one embodiment, the liquid formulation is a pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.
In one embodiment, the liquid antibody formulation of the invention comprises:
(i) about 1-200mg/ml of an anti-CD 47/PD-L1 bispecific antibody protein;
(ii) about 1-30mM histidine and/or histidine hydrochloride;
(iii) about 50-500mM of sucrose, arginine and/or arginine hydrochloride, and
(iv) about 0.1-1mg/ml polysorbate 80;
optionally, the liquid formulation further comprises 0.002-0.2 mg/ml metal chelating agent (e.g., EDTA);
wherein the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
In a preferred embodiment, the liquid antibody formulation of the invention comprises:
(i) about 50-150mg/ml of an anti-CD 47/PD-L1 bispecific antibody protein;
(ii) about 3-25mM histidine and/or histidine hydrochloride;
(iii) about 150 mM of sucrose, arginine and/or arginine hydrochloride, and
(iv) about 0.2-0.8mg/ml polysorbate 80;
optionally, the liquid formulation further comprises about 0.01-0.1mg/ml metal chelator (e.g., EDTA);
wherein the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
In a preferred embodiment, the liquid antibody formulation of the invention comprises
(i) About 100mg/ml of an anti-CD 47/PD-L1 bispecific antibody protein;
(ii) about 20mM histidine;
(iii) about 180mM arginine hydrochloride, and
(iv) about 0.2mg/ml polysorbate 80;
optionally, the liquid formulation further comprises a metal chelator (e.g., EDTA), such as about 0.02mg/ml EDTA;
wherein the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
In a preferred embodiment, the liquid antibody formulation of the invention comprises
(i) About 100mg/ml of an anti-CD 47/PD-L1 bispecific antibody protein;
(ii) about 20mM histidine;
(iii) about 100mM arginine hydrochloride and 4% sucrose, and
(iv) about 0.2mg/ml polysorbate 80;
optionally, the liquid formulation further comprises a metal chelator (e.g., EDTA), such as about 0.02mg/ml EDTA;
wherein the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
In a preferred embodiment, the liquid antibody formulation of the invention comprises
(i) About 100mg/ml of an anti-CD 47/PD-L1 bispecific antibody protein;
(ii) about 2.52mg/ml histidine and about 0.79mg/ml histidine hydrochloride;
(iii) about 37.92mg/ml arginine hydrochloride, and
(iv) about 0.5mg/ml polysorbate 80;
optionally, the liquid formulation further comprises a metal chelator (e.g., EDTA), such as about 0.02mg/ml EDTA;
wherein the pH of the liquid formulation is about 6.4-7.0, preferably about 6.5.
In another aspect, the present invention provides ー solid antibody preparations obtained by subjecting the liquid antibody preparation of the present invention to a curing treatment. The solidification treatment is carried out by, for example, crystallization, spray drying, or freeze drying. In a preferred embodiment, the solid antibody formulation is, for example, in the form of a lyophilized powder injection. Solid antibody formulations can be reconstituted in a suitable vehicle to form reconstituted formulations of the invention prior to use. The reconstituted formulation is also a liquid antibody formulation of the invention. In one embodiment, the suitable vehicle is selected from water for injection, organic solvents for injection, including but not limited to oil for injection, ethanol, propylene glycol, and the like, or combinations thereof.
The liquid formulations of the present invention can be stably stored for a long period of time, for example, at least 24 months or longer. In one embodiment, the liquid formulation of the present invention may be stable upon storage at about-80 ℃ to about 45 ℃, e.g., -80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃ for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer.
In one embodiment, the liquid formulation of the present invention is stable for storage for at least 24 months. In yet another embodiment, the liquid formulation of the present invention is stable at least 40 ℃. In yet another embodiment, the liquid formulation of the present invention remains stable for at least 12 months, preferably at least 24 months at about 2 ℃ to 8 ℃. In one embodiment, the liquid formulation of the invention remains stable for at least 3 months, preferably at least 6 months, at room temperature or, for example, about 25 ℃. In yet another embodiment, the liquid formulation of the present invention remains stable for at least 1 month at about 40 ℃.
In one embodiment, the stability of the formulation after storage can be indicated by detecting changes in the appearance, visible foreign matter, protein content, purity, and/or charge variants of the formulation. In one embodiment, the stability of the liquid formulations of the present invention may be tested in a high temperature stress test, for example after storage at 40 ℃ ± 2 ℃ for at least 1 week, 2 weeks or preferably 1 month, or after storage at 25 ℃ ± 2 ℃ for at least 1 month or 2 months.
In one embodiment, the stability of the liquid formulations of the present invention is visually inspected after storage, wherein the liquid formulations of the present invention remain clear to slightly opalescent in appearance, as colorless to pale yellow liquids, and free of foreign objects. In one embodiment, no visible foreign matter is present in the formulation upon visual inspection under a clarity detector. In one embodiment, the stability of the liquid formulations of the invention is checked after storage by determining the change in protein content, wherein the rate of change in protein content is not more than 20%, preferably not more than 10%, such as 7-8%, preferably not more than 5%, relative to the initial value on day 0 of storage, for example by ultraviolet spectrophotometry (UV). In one embodiment, the stability of the liquid formulations of the invention is checked after storage by determining the turbidity change of the liquid formulations of the invention, e.g. by OD350mmThe method detects that the variation value is not more than 0.04, more preferably not more than 0.03, and more preferably not more than 0.02, relative to the initial value on day 0 of storage. In one embodiment, the stability of the liquid formulations of the invention is checked after storage by determining the change in purity of the liquid formulations of the invention, whichBy size exclusion high performance liquid chromatography (SEC-HPLC), the monomer purity does not vary by more than 10%, such as by more than 5%, 4%, 3%, such as 1-2%, preferably by more than 1%, from the initial value on day 0 of storage. In one embodiment, the stability of the liquid formulation of the invention is checked after storage by determining the change in purity of the liquid formulation of the invention, wherein the change in monomer purity is reduced by no more than 10%, such as no more than 9.5%, 8.5%, 7.5%, 6.5%, by non-reduced and/or reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method. In one embodiment, the stability of the liquid formulation of the invention is tested after storage by imaged capillary isoelectric focusing electrophoresis (iCIEF), wherein the sum of the change values of the charge variants (main component, acidic component and basic component) of the antibody does not exceed 50%, such as not more than 45%, 40%, 35%, 30%, 25%, relative to the initial value on day 0 of storage. In one embodiment, the stability of the liquid formulation of the invention is tested after storage by cation exchange high performance liquid chromatography (CEX-HPLC method), wherein the sum of the change values of the charge variants (main component, acidic component and basic component) of the antibody does not exceed 40%, such as 38%, 36%, 34%, 32%, 30%, relative to the initial value on day 0 of storage.
In one embodiment, the formulation is stable after storage, e.g. after storage at 2-8 ℃ for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40 ℃ ± 2 ℃ for 1 month, preferably having one or more of the following characteristics:
(i) the formulations have a purity of greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99% as measured by SEC-HPLC;
(ii) the formulations have a purity of greater than 85%, preferably greater than 86%, 87%, 88%, 89% as measured by the reduced or non-reduced CE-SDS method;
(iii) the sum of the variation values of the components (main, acidic and basic) of the anti-CD 47/PD-L1 bispecific antibody protein in the formulation, as measured by the iCIEF method, relative to the initial value on day 0 of storage, is not more than 50%, e.g. not more than 45%, 40%, 35%, 30%, 25%;
(iv) the sum of the variation values of the components (main, acidic and basic) of the anti-CD 47/PD-L1 bispecific antibody protein in the formulation, as measured by the CEX-HPLC method, relative to the initial value on day 0 of storage, is not more than 40%, e.g. not more than 38%, 36%, 34%, 32%, 30%;
(v) the relative binding activity of the anti-CD 47/PD-L1 bispecific antibody protein in the formulation, as measured by ELISA, is 70% to 130%, e.g., 70%, 80%, 90%, 100%, 110%, 120%, 130%, relative to the initial value on day 0 of storage.
In one aspect, the invention provides a delivery device comprising a liquid antibody formulation or a solid antibody formulation of the invention. In one embodiment, the delivery device of the invention is provided in the form of a pre-filled syringe comprising a liquid or solid antibody formulation of the invention, e.g. for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.
In yet another aspect, the invention provides a method of delivering an anti-CD 47/PD-L1 bispecific antibody protein to a subject, e.g., a mammal, comprising the step of administering to the subject a liquid antibody formulation or a solid antibody formulation of the invention, e.g., by a delivery device using a pre-filled syringe.
In a further aspect, the invention provides the use of a liquid or solid antibody formulation of the invention for the preparation of a delivery device (e.g., a pre-filled syringe) or medicament for the treatment, prevention or delay of a condition associated with sirpa/CD 47 signaling pathway and PD1/PD-L1 signaling pathway, such as various hematological and solid tumors, including but not limited to Acute Myeloid Leukemia (AML), chronic myeloid leukemia, Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), lymphoma, breast cancer, gastric cancer, lung cancer, esophageal cancer, intestinal cancer, ovarian cancer, cervical cancer, renal cancer, pancreatic cancer, bladder cancer, glioma, melanoma and other solid tumors in a subject; autoimmune diseases and inflammatory disorders, for example, allergic asthma or ulcerative colitis; cell or tissue or organ transplant rejection, including non-human tissue transplant (xenograft) rejection.
Other embodiments of the invention will be apparent by reference to the detailed description that follows.
Brief Description of Drawings
The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Figure 1 illustrates the structure of an anti-CD 47/PD-L1 bispecific antibody in which a first polypeptide chain is paired with a second polypeptide chain to form a first antigen binding site, a third polypeptide chain comprises a single domain second antigen binding site and a single domain third antigen binding site, and there is a flexible connecting peptide between the single domain second antigen binding site and the single domain third antigen binding site of the third polypeptide chain.
FIG. 2 shows a trend graph of protein purity in each sample as determined by SEC-HPLC after various time periods at pH 6.4, 6.5, 6.8 and 7.0 at 40 ℃. + -. 2 ℃ for anti-CD 47/PD-L1 bispecific antibody formulations. T0 on the abscissa of the graph represents day 0; 2W represents 2 weeks; 1M represents 1 month.
FIG. 3 shows a trend graph of protein purity in each sample determined by the non-reduced CE-SDS method after the anti-CD 47/PD-L1 bispecific antibody formulation was left at pH 6.4, 6.5, 6.8 and 7.0 at 40 ℃. + -. 2 ℃ for various periods of time. T0 on the abscissa of the graph represents day 0; 2W represents 2 weeks; 1M represents 1 month.
FIG. 4 shows a graph of the trend of charge variant principal components in each sample as determined by the iCIEF method for anti-CD 47/PD-L1 bispecific antibody formulations after standing at pH 6.4, 6.5, 6.8, and 7.0 at 40 ℃. + -. 2 ℃ for various periods of time. T0 on the abscissa of the graph represents day 0; 2W represents 2 weeks; 1M represents 1 month.
FIG. 5 shows graphs of main peak purity over time as determined by SEC-HPLC after storage of anti-CD 47/PD-L1 bispecific antibody formulations (recipes 2-5) with different stabilizers at 40 ℃. + -. 2 ℃ for 0 day, 1 week, 2 weeks, and 4 weeks. T0 on the abscissa of the graph represents day 0; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks.
FIG. 6 shows the main peak purity as a function of time, measured by the non-reduced CE-SDS method, after storage of anti-CD 47/PD-L1 bispecific antibody formulations (recipes 2-5) at 40 ℃. + -. 2 ℃ for 0 day, 1 week, 2 weeks, and 4 weeks. T0 on the abscissa of the graph represents day 0; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks.
FIG. 7 shows graphs of charge variant principal components as a function of time, as determined by the iCIEF method, after storage of anti-CD 47/PD-L1 bispecific antibody formulations (recipes 2-5) with different stabilizers at 40 ℃. + -. 2 ℃ for 0 day, 1 week, 2 weeks, and 4 weeks. T0 on the abscissa of the graph represents day 0; 1W represents 1 week; 2W represents 2 weeks; 4W represents 4 weeks.
Detailed Description
Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology and experimental conditions set forth herein as such may vary. In addition, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.
The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.
The term "and/or" when used to connect two or more selectable items should be understood to mean either one of the selectable items or any two or more of the selectable items.
As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.
The term "antibody" is used herein in the broadest sense to refer to a protein that comprises an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to, three-chain antibodies, intact antibodies, and antigen-binding fragments of antibodies.
The terms "whole antibody", "full-length antibody", "whole antibody" and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions (as Complementarity Determining Regions (CDRs) with more conserved regions (as Framework Regions (FRs)) interposed between each VH and VL consisting of three CDRs and 4 FRs arranged in the order from amino to carboxyl, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. the constant regions are not directly involved in the binding of antibodies to antigens, but exhibit multiple effector functions.
The term "antibody preparation" refers to a preparation in a form that allows the biological activity of an antibody as an active ingredient to be exerted effectively, and that does not contain other components having unacceptable toxicity to the subject to which the preparation is to be administered. Such antibody preparations are generally sterile. Typically, pharmaceutically acceptable excipients are included in the antibody formulation. A "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a subject mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration and may, for example, be acceptable for injection.
The term "anti-CD 47/PD-L1 bispecific antibody preparation", also referred to herein simply as "the antibody preparation of the invention", means a preparation comprising an anti-CD 47/PD-L1 bispecific antibody protein as an active ingredient and comprising pharmaceutically acceptable excipients. The anti-CD 47/PD-L1 bispecific antibody protein as an active ingredient is suitable for therapeutic or prophylactic administration to human or non-human animals after combining the anti-CD 47/PD-L1 bispecific antibody protein with pharmaceutically acceptable excipients. The antibody formulations of the invention may be prepared, for example, as liquid formulations in aqueous form, e.g., ready-to-use prefilled syringes, or as lyophilized formulations for reconstitution (i.e., reconstitution) by dissolution and/or suspension in a physiologically acceptable solution immediately prior to use. In some embodiments, the anti-CD 47/PD-L1 bispecific antibody protein formulation is in the form of a liquid formulation.
A "stable" antibody formulation is one in which the antibodies in the formulation retain an acceptable degree of physical and/or chemical stability after storage under specified conditions. An antibody preparation is considered "stable" although an antibody contained in the antibody preparation may not 100% maintain its chemical structure after a particular time of storage, typically maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function after a particular time of storage. In some particular embodiments, the anti-CD 47/PD-L1 bispecific antibody protein formulations of the present invention exhibit as little as undetectable antibody aggregation or degradation or chemical modification during manufacture, transport, and long term storage, thereby exhibiting high stability with little or even no loss of biological activity of the anti-CD 47/PD-L1 bispecific antibody protein. In some embodiments, the anti-CD 47/PD-L1 bispecific antibody protein formulations of the present invention substantially retain their physical and chemical stability after storage. Preferably, the liquid formulations of the present invention may be stable for at least 1 month at room temperature or at 40 ℃ and/or at 2-8 ℃ for at least 24 months.
A variety of analytical techniques are known in the art for determining the stability of proteins, see, e.g., Peptide and Protein Drug Delivery,247- & 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs (1991) and Jones, A.Adv.drug Delivery Rev.10:29-90 (1993). Stability can be measured at selected temperatures and selected storage times. For example, the storage time may be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test may be used. In some embodiments, the stability test is performed by performing various stress tests on the antibody formulation. These tests may represent extreme conditions that a formulated antibody preparation may encounter during manufacturing, storage or transportation, and may also represent conditions that may accelerate the instability of the antibodies in the antibody preparation during non-manufacturing, storage or transportation. For example, a formulated anti-CD 47/PD-L1 bispecific antibody protein formulation can be filled into glass vials to examine antibody stability under high temperature stress.
The formulation does not show aggregation, precipitation, turbidity and/or denaturation after a period of storage; or exhibit very little aggregation, precipitation, turbidity, and/or denaturation, the antibody can be considered to "maintain its physical stability" in the formulation. Safety issues arise due to aggregation of antibodies in the formulation which can potentially lead to increased immune responses in the patient. Therefore, there is a need to minimize or prevent aggregation of antibodies in a formulation. Light scattering methods may be used to determine visible aggregates in the formulation. SEC can be used to determine soluble aggregates in the formulation. In addition, the appearance, color and/or clarity of the formulation can be visually inspected, or by OD350nmThe stability of the formulation is indicated by measuring the turbidity of the formulation or by measuring the purity of the formulation by the non-reduced CE-SDS method. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomer in the formulation after storage at a particular temperature for a particular time, wherein the higher the percentage of antibody monomer in the formulation, the higher the stability of the formulation.
An "acceptable degree" of physical stability may mean that at least about 92% of the anti-CD 47/PD-L1 bispecific antibody protein monomers are detected in the formulation after storage for a specified time at a specified temperature. In some embodiments, an acceptable degree of physical stability after storage at a particular temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more represents at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-CD 47/PD-L1 bispecific antibody protein monomer. When assessing physical stability, the particular temperature at which the pharmaceutical formulation is stored may be any temperature from about-80 ℃ to about 45 ℃, e.g., about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ℃ to 8 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 37 ℃, about 40 ℃, about 42 ℃, or about 45 ℃. For example, a pharmaceutical formulation is considered stable if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-CD 47/PD-L1 bispecific antibody protein monomer is detected after 1 month or 4 weeks of storage at about 40 ℃ ± 2 ℃. A pharmaceutical formulation is considered stable if after 2 months of storage at about 25 ℃, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-CD 47/PD-L1 bispecific antibody protein monomer is detected. A pharmaceutical formulation is considered stable if, after 9 months of storage at about 5 ℃, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-CD 47/PD-L1 bispecific antibody protein monomer is detected.
An antibody may be considered to "retain its chemical stability" in a formulation if the antibody in the formulation does not show a significant chemical change after a period of storage. Most chemical instability results from the formation of covalently modified forms of antibodies (e.g., charge variants of antibodies). Basic variants can be formed, for example, by aspartic acid isomerization, N-and C-terminal modifications; acidic variants can be produced by deamidation, sialylation and saccharification. Chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody. For example, charge variants of antibodies in a preparation can be detected by cation exchange Chromatography (CEX) or imaging capillary isoelectric focusing (iCIEF). In one embodiment, the stability of the formulation is measured by determining the value of the percent change in charge variant of the antibody in the formulation after storage at a particular temperature for a particular time, wherein the smaller the change value, the higher the stability of the formulation.
An "acceptable degree" of chemical stability may mean that the percentage change in the charge variant (e.g., the major component or acidic component or basic component) in the formulation after storage for a specified time at a specified temperature does not exceed a value of 30%, e.g., 20%. In some embodiments, an acceptable degree of chemical stability may be manifested as a percent change in the acidic component charge variant of no more than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% after storage at a particular temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or more. When assessing chemical stability, the temperature at which the pharmaceutical formulation is stored may be any temperature from about-80 ℃ to about 45 ℃, e.g., at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ℃ to 8 ℃, about 5 ℃, about 25 ℃, or about 45 ℃. For example, a pharmaceutical formulation may be considered stable if the percent change in acidic component charge variants after 24 months of storage at 5 ℃ has a value of less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. A pharmaceutical formulation may also be considered stable if the percent change in the acidic component charge variant after storage at 25 ℃ for 2 months has a value of less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%. A pharmaceutical formulation may also be considered stable if the percent change in acidic component charge variants after 1 month storage at 40 ℃ has a value of less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.
The term "lyophilized formulation" refers to a composition obtained or obtainable by a freeze-drying process of a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.
The term "reconstituted formulation" refers to a liquid formulation resulting from dissolving and/or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.
The term "room temperature" as used herein means a temperature of from 15 ℃ to 30 ℃, preferably from 20 ℃ to 27 ℃, more preferably 25 ℃.
"stress conditions" refer to environments that are chemically and/or physically hostile to an antibody protein, which may result in unacceptable destabilization of the antibody protein. By "high temperature stress" is meant that the antibody formulation is stored at room temperature or even higher (e.g., 40 ℃. + -. 2 ℃) for a period of time. The stability of the antibody formulations can be checked by high temperature stress accelerated tests.
As used herein, the term "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular (subepithelial), intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, the stable anti-CD 47/PD-L1 bispecific antibody protein formulation of the present invention is administered parenterally to a subject. In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.
I. Antibody formulations
The present invention provides a stable liquid antibody formulation comprising (i) an anti-CD 47/PD-L1 bispecific antibody protein, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, optionally further comprising (v) other excipients, the antibody formulation having a pH of about 6.4-7.0. In a preferred embodiment, the liquid antibody formulation of the invention is in the form of an injectable formulation.
(i) anti-CD 47/PD-L1 bispecific antibody protein
The "anti-CD 47/PD-L1 bispecific antibody protein" in the antibody preparation of the invention is a three chain antibody comprising a VH/VL pair specifically binding CD47 on a first polypeptide chain and a second polypeptide chain as a first antigen-binding site, and a first VHH specifically binding PD-L1 on a third polypeptide chain as a single domain second antigen-binding site and a second VHH as a single domain third antigen-binding site; or a VH/VL pair on a first and second polypeptide chain that specifically binds PD-L1 as a first antigen binding site and a first VHH on a third polypeptide chain that specifically binds CD47 as a single domain second antigen binding site and a second VHH as a single domain third antigen binding site. The anti-CD 47/PD-L1 bispecific antibody protein can be at least about 107M-1Preferably about 108M-1And more preferably about 109M-1Or stronger, and is capable of binding to CD47 with an affinity constant of at least about 107M-1Preferably about 108M-1And more preferably about 109M-1Or a stronger affinity constant, to PD-L1, such that the antibody can be used as a therapeutic and/or prophylactic agent for bispecific targeting of a CD47 molecule and a PD-L1 molecule.
For the VH/VL pair that specifically binds PD-L1 or CD47, comprising sequences derived from 6 CDRs of any anti-PD-L1 antibody reported in the prior art and an anti-PD-L1 antibody VH/VL pair developed in the future or having one, two, three, four, five, six, or more amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs of the 6 CDRs; or a sequence derived from any of the anti-CD 47 antibodies reported in the prior art and the 6 CDRs of a future developed anti-CD 47 antibody VH/VL pair or having one, two, three, four, five, six, or more amino acid changes (e.g., amino acid substitutions or deletions) with one or more of the 6 CDRs.
For the first VHH and the second VHH that specifically bind PD-L1 or CD47, they are both derived from the heavy chain variable domain of an antibody that naturally lacks a light chain (such as the heavy chain variable domain of a heavy chain antibody naturally occurring in a species in the family Camelidae (Camelidae)). The first VHH and the second VHH may be the same or different. The first VHH and the second VHH may be derived from antibodies raised in species in the family camelidae (e.g. camel, alpaca, dromedary, llama and guanaco). Other species than camelidae may also produce heavy chain antibodies naturally lacking the light chain, such VHHs are also within the scope of the antibody protein of the invention. The first and second VHHs comprise sequences derived from 3 CDRs of any anti-PD-L1 antibody reported in the prior art and an anti-PD-L1 antibody VHH developed in the future or having one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs of the 3 CDRs; or a sequence derived from 3 CDRs of any of the anti-CD 47 antibodies reported in the prior art and the anti-CD 47 antibody VHH developed in the future or having one, two, three, four, five, six, or more amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs of the 3 CDRs.
In one embodiment, the VH/VL pair specifically binding CD47 on said first and second polypeptide chains of the anti-CD 47/PD-L1 bispecific antibody protein comprises VH CDR1 as shown derived from GSIEHYYWS (SEQ ID NO:3), VH CDR2 as shown by YIYYSGSTNYNPSLKS (SEQ ID NO:4), VH CDR3 as shown by ARGKTGSAA (SEQ ID NO:5), VL CDR1 as shown by RASQGISRWLA (SEQ ID NO:10), VL CDR2 and QQTVSFPIT as shown by AASSLQS (SEQ ID NO:11) (SEQ ID NO:12), or a sequence having one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs of said 6 CDRs reported by chinese patent application No. CN201710759828.9 for the anti-CD 47 antibody ADI-29341 (SEQ ID NO: 3).
In one embodiment, the first VHH and the second VHH on the third polypeptide chain of the anti-CD 47/PD-L1 bispecific antibody protein that specifically bind to PD-L1 each comprise CDR1 as shown in AYTISRNSMG (SEQ ID NO:17), CDR2 as shown in IESDGST (SEQ ID NO:18) and CDR3 as shown in AAPKVGLGPRTALGHLAFMTLPALNY (SEQ ID NO:19), or a sequence with one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) to one or more CDRs of the 3 CDRs.
The term "CDR" or "complementarity determining region" or "CDR region" (used interchangeably herein with hypervariable region "HVR") is the region of amino acids in an antibody variable region that is primarily responsible for binding to an epitope of an antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. Various protocols are known in the art for determining the CDR sequences of a given VH or VL or VHH amino acid sequence. For example, Kabat Complementarity Determining Regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). And Chothia refers to the position of the structural loops (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). The AbM HVR is a compromise between the Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "Contact" HVR is based on an analysis of the complex crystal structure available. HVRs can also be determined based on having the same Kabat numbered position as a reference CDR sequence (e.g., exemplary CDRs disclosed herein).
The amino acid changes, e.g., amino acid substitutions, are preferably conservative amino acid substitutions. "conservative amino acid substitution" refers to an amino acid change that results in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. In any embodiment of the invention, in a preferred aspect, the conservatively substituted residue is from the following conservative substitution representative of a, preferably the preferred replacement residues shown in table a.
TABLE A
| Original residues | Exemplary substitutions | Preferred conservative amino acid substitutions |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Asp；Lys；Arg | Gln |
| Asp(D) | Glu；Asn | Glu |
| Cys(C) | Ser；Ala | Ser |
| Gln(Q) | Asn；Glu | Asn |
| Glu(E) | Asp；Gln | Asp |
| Gly(G) | Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu; val; met; ala; phe; norleucine | Leu |
| Leu(L) | Norleucine; ile; val; met; ala; phe (Phe) | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Trp；Leu；Val；Ile；Ala；Tyr | Tyr |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Val；Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile; leu; met; phe; ala; norleucine | Leu |
In one embodiment, the VH/VL pair that specifically binds CD47 on said first and second polypeptide chains of the anti-CD 47/PD-L1 bispecific antibody protein comprises a heavy chain variable region sequence/light chain variable region sequence derived from SEQ ID NO:2/9 of anti-CD 47 antibody ADI-29341, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said heavy chain variable region sequence/light chain variable region sequence.
In one embodiment, the first VHH and the second VHH on said third polypeptide chain of the anti-CD 47/PD-L1 bispecific antibody protein that specifically bind to PD-L1 comprise the amino acid sequences set forth in SEQ ID NO:15 and/or SEQ ID NO:16, or sequences that are substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto.
The type of heavy chain constant region of the immunoglobulin in the first and third polypeptide chains of the anti-CD 47/PD-L1 bispecific antibody protein is not particularly limited, preferably is that of an IgG1, IgG2, or IgG4 immunoglobulin, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto. More preferably, the heavy chain constant region is a heavy chain constant region of a human IgG1 immunoglobulin, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) thereto.
In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein comprises a heavy chain constant region used in IgG4 (e.g., human IgG 4). In yet another embodiment, the anti-CD 47/PD-L1 bispecific antibody protein comprises a heavy chain constant region for IgG1 (e.g., human IgG 1). For example, the first and third polypeptide chains of a three chain antibody comprise in their Fc domains a hinge region with "CPPC" amino acid residues, respectively, and/or Y349C and S354C, respectively (according to Kabat' EU numbering "), whereby the first and third polypeptide chains form inter-chain disulfide bonds in the Fc region, thereby stabilizing the correct pairing of the first and third polypeptide chains.
In one embodiment, the first polypeptide chain and/or the third polypeptide chain of the anti-CD 47/PD-L1 bispecific antibody protein comprises an amino acid mutation in the Fc domain that affects antibody effector function. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the amino acid mutation is present in the CH2 domain of the Fc region, e.g., the anti-CD 47/PD-L1 bispecific antibody protein comprises an amino acid substitution at positions 234 and 235 (EU numbering) of the first polypeptide chain and/or the third polypeptide chain. In a specific embodiment, the amino acid substitutions are L234A and L235A (also referred to as "LALA mutations").
In yet another embodiment, the second polypeptide chain of the anti-CD 47/PD-L1 bispecific antibody protein comprises a kappa light chain constant region or a lambda light chain constant region, e.g., a human kappa light chain constant region or a human lambda light chain constant region.
In one embodiment, the Fc domains of each of the first and third polypeptide chains of the anti-CD 47/PD-L1 bispecific antibody protein comprise a bulge ("knob") or a hole ("hole"), respectively, and said bulge or hole in the Fc domain of the first polypeptide chain can be placed in said hole or bulge, respectively, in the Fc domain of the third polypeptide chain, whereby said first and third polypeptide chains form a stable association of "knob-in-hole" with each other. In one embodiment, the amino acid substitution T366W is included in one of said first and third polypeptide chains and the amino acid substitutions T366S, L368A and Y407V (EU numbering) are included in the other of said first and third polypeptide chains. Whereby the protuberance in one strand is able to be placed in the cavity in the other strand, facilitating proper pairing of the first polypeptide chain and the third polypeptide chain.
In one embodiment, the immunoglobulin CH1 domain and the CL domain of the first and second polypeptide chains of the anti-CD 47/PD-L1 bispecific antibody protein comprise a bulge or a hole, respectively, and said bulge or hole in the CH1 domain can be placed in said hole or bulge, respectively, in the CL domain, such that said first and second polypeptide chains also form a stable association of "knot-in" with each other.
In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein comprises a first polypeptide chain as set forth in SEQ ID NO:1, a second polypeptide chain as set forth in SEQ ID NO:8, and a third polypeptide chain as set forth in SEQ ID NO:14, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any one of said sequences.
In yet another embodiment, an anti-CD 47/PD-L1 bispecific antibody protein comprises a first polypeptide chain set forth in SEQ ID NO:1, a second polypeptide chain set forth in SEQ ID NO:8, and a third polypeptide chain set forth in SEQ ID NO:22, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any one of the recited sequences.
As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window. The "percent sequence identity" can be calculated by: the two optimally aligned sequences are compared over a comparison window, the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence identity. Optimal alignment for determining percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence.
The anti-CD 47/PD-L1 bispecific antibody protein in the antibody preparation can be combined with PD-L1 and CD47 proteins at the same time, and maintains the affinity constant of each parent antibody, so that the SIRPa/CD 47 signal conduction pathway can be blocked and the PD1/PD-L1 signal conduction pathway can be blocked, and the antibody preparation can be used for treating, preventing or delaying various diseases or symptoms related to the SIRPa/CD 47 signal conduction pathway and/or the PD1/PD-L1 signal conduction pathway.
In a preferred embodiment, the anti-CD 47/PD-L1 bispecific antibody protein of the invention is a recombinant anti-CD 47/PD-L1 bispecific antibody protein disclosed in PCT application PCT/CN2018/123886 (application date: 2018, 26.12), having a first polypeptide chain of SEQ ID No. 1, a second polypeptide chain of SEQ ID No. 8, and a third polypeptide chain of SEQ ID No. 14. In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein is produced by recombinant expression from HEK293 cells or CHO cells and purified. Preferably, the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity. Administration of an anti-CD 47/PD-L1 bispecific antibody to tumor-bearing mice generated by inoculation of NOD-SCID mice with Raji-PD-L1 cells showed that administration of an anti-CD 47/PD-L1 bispecific antibody has significantly improved anti-tumor activity, which may result in a tumor growth inhibition rate of about 90% or more, e.g., 100%, compared to administration of an anti-CD 47 monoclonal antibody and an anti-PD-L1 monoclonal antibody; and/or the tumor disappearance rate reaches more than 50 percent. Furthermore, the anti-CD 47/PD-L1 bispecific antibody also exhibits significantly reduced hemagglutination and will therefore have significantly reduced side effects in clinical treatment.
The amount of anti-CD 47/PD-L1 bispecific antibody protein included in an antibody formulation of the invention can vary with the particular characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is used. In some embodiments, the antibody formulation is a liquid formulation, which may contain about 5-150mg/mL, e.g., about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150mg/mL of the anti-CD 47/PD-L1 bispecific antibody protein.
(ii) Buffering agent
Buffers are agents that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffering agent used in the formulations of the present invention may control the pH of the formulations of the present invention in the pH range of about 6.4-7.0, for example, a pH of about 6.5. In some specific embodiments, an antibody formulation of the invention has a pH of about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.
In some embodiments, the buffering agent used in the formulations of the present invention is selected from histidine, histidine hydrochloride, and combinations thereof. In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 1-30 mM. In one embodiment, the buffer in a liquid antibody formulation of the invention is at a concentration of about 5-25mM, e.g., about 5, 10, 15, 20, 25 mM.
In one embodiment, the buffer used in the formulation of the present invention is a combination of about 16.3mM histidine and about 3.77mM histidine hydrochloride.
(iii) Stabilizer
Suitable stabilizers for use in the present invention may be selected from sugars, polyols and amino acids and combinations thereof. Examples of sugars that act as stabilizers include, but are not limited to, sucrose and trehalose. Examples of polyols as stabilizers include, but are not limited to, sorbitol. For amino acids as stabilizers, but not limited to, arginine hydrochloride. In some embodiments, the stabilizing agent is present in the liquid formulation of the invention at a concentration of about 50-500mM, more preferably about 100-400mM, e.g., about 100, 150, 200, 250, 300, 350, 400 mM.
In one embodiment, the liquid formulation of the present invention comprises sucrose as a stabilizer. The amount of sucrose in the liquid formulation of the present invention may be about 50-250mM, preferably about 100 and 200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
In one embodiment, the liquid formulation of the present invention comprises arginine and/or arginine hydrochloride as a stabilizer. The amount of arginine and/or arginine hydrochloride in the liquid formulation of the present invention may be about 50-250mM, preferably about 100-200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
In one embodiment, the liquid formulation of the present invention comprises a combination of sucrose, arginine and/or arginine hydrochloride as a stabilizer. In this combination, sucrose may be present in an amount of about 50-250mM, preferably about 100 and 200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM). In this combination, arginine and/or arginine hydrochloride may be present in an amount of about 50-250mM, preferably about 100-200mM (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM).
(iv) Surface active agent
As used herein, the term "surfactant" refers to an organic substance having an amphiphilic structure; that is, they are composed of groups of opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group.
In one embodiment, the surfactant in the liquid formulation of the present invention is a non-ionic surfactant, for example, an alkyl poly (ethylene oxide). Specific nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; pluronic, and the like. In a preferred embodiment, polysorbate-80 is included in the liquid formulation of the present invention as a surfactant.
The amount of surfactant contained in an antibody formulation of the invention may vary with the particular intended characteristics of the formulation, the particular environment, and the particular purpose for which the formulation is used. In preferred embodiments, the formulation may contain about 0.1-1mg/ml, preferably about 0.2-0.8mg/ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8mg/ml of a polysorbate-based surfactant (e.g., polysorbate-80).
(v) Other excipients
Other excipients may or may not be included in the antibody liquid formulations of the present invention.
In one embodiment, the antibody liquid formulation of the invention comprises a metal chelator (e.g., EDTA or a salt thereof) as an excipient. In another embodiment, the antibody liquid formulation of the invention does not comprise a metal chelator (e.g., EDTA or a salt thereof). In one embodiment, a liquid formulation of the antibody of the invention with the addition of a metal chelator (e.g., EDTA or a salt thereof) has greater stability than a corresponding formulation without the addition of a metal chelator (e.g., EDTA or a salt thereof).
Other excipients may also be used in the formulations of the present invention for other considerations. Such excipients include, for example, flavoring agents, antimicrobial agents, sweetening agents, antistatic agents, antioxidants, gelatin, and the like. These and additional known Pharmaceutical Excipients and/or additives suitable for use in The formulations of The present invention are well known in The art, for example, as listed in "The Handbook of Pharmaceutical Excipients, 4 th edition, edited by Rowe et al, American Pharmaceuticals Association (2003); and Remington the Science and Practice of Pharmacy, 21 st edition, eds. Gennaro, Lippincott Williams & Wilkins (2005) ".
Preparation of the formulations
The present invention provides stable formulations comprising an anti-CD 47/PD-L1 bispecific antibody protein. The anti-CD 47/PD-L1 bispecific antibody proteins used in the formulations of the invention can be prepared using techniques known in the art for the production of antibodies. For example, an anti-CD 47/PD-L1 bispecific antibody protein can be recombinantly produced. In a preferred embodiment, the anti-CD 47/PD-L1 bispecific antibody protein of the invention is prepared by recombinant expression in HEK293 cells or CHO cells, e.g. as described in PCT/CN2018/123886, an anti-CD 47/PD-L1 bispecific antibody protein is recombinantly prepared.
The use of antibodies as active ingredients of pharmaceuticals is now widespread. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al (knowledge purification of chromatography steps for purification of monomeric antibodies, Biotechnology and Bioengineering 99(2008) 599-613.) describe a method of antibody three-column purification using ion exchange chromatography (anionic IEX and/or cationic CEX chromatography) after a protein A capture step. Kelley et al (Weak chromatography for exchange purification of monomeric antibiotics, Biotechnology and Bioengineering 101(2008) 553-566) describe a two-column purification method in which a weakly-partitioning anion exchange resin is used after protein A affinity chromatography.
In general, recombinantly produced antibodies can be purified using conventional purification methods to provide a drug substance with sufficient reproducibility and moderate purity for the formulation of antibody preparations. For example, after secretion of the antibody from the recombinant expression cells into the culture medium, the supernatant from the expression system can be concentrated using a commercially available protein concentration filter, such as the Amicon's ultrafiltration device. Thereafter, purification of the antibody can be performed using means such as chromatography, dialysis, and affinity purification. Protein a is adapted as an affinity ligand for the purification of antibodies of the IgG1, IgG2 and IgG4 types. Other antibody purification methods, such as ion exchange chromatography, may also be used. After obtaining an antibody of sufficient purity, preparations comprising the antibody can be prepared according to methods known in the art.
For example, the preparation can be carried out by the following steps: (1) after fermentation is finished, centrifuging and clarifying the fermentation liquor to remove impurities such as cells and the like so as to obtain a supernatant; (2) capture of antibodies using affinity chromatography (e.g., protein a columns with specific affinity for IgG1, IgG2, and IgG 4-type antibodies); (3) performing virus inactivation; (4) refining and purifying (CEX cation exchange chromatography can be adopted generally) to remove impurities in the protein; (4) virus filtration (to reduce virus titer by, e.g., more than 4log 10); (5) ultrafiltration/diafiltration (which may be used to displace the protein in formulation buffer to facilitate its stability and concentrate to a suitable concentration for injection). See, e.g., B.Minow, P.Rogge, K.Thompson, BioProcess International, Vol.10, No.6,2012, pp.48-57.
Method for analyzing preparation
During storage of an antibody preparation, the antibodies may aggregate, degrade, or chemically modify, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), as well as aggregates and fragments, etc., thereby affecting the quality of the antibody preparation. Therefore, monitoring of the stability of the antibody formulation is necessary.
Various methods are known in the art for testing the stability of antibody formulations. For example, the purity of the antibody preparation can be analyzed and the aggregation level of the antibody can be evaluated by methods such as reduced CE-SDS, non-reduced CE-SDS and SEC-HPLC; charge variants in antibody preparations can be analyzed by capillary isoelectric focusing electrophoresis (cIEF), imaged capillary isoelectric focusing electrophoresis (iCIEF), ion exchange chromatography (IEX), and the like. In addition, the stability of the formulation can be rapidly judged by visually inspecting the appearance of the formulation. OD may also be used350nmThe method measures the turbidity change of the formulation and gives information about the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) may be used to detect changes in protein content in the formulation.
The non-reduced CE-SDS method is a method for determining the purity of monoclonal antibodies by using a capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge caused by SDS binding, which is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on the size or hydrodynamic radius of the molecules can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies. Generally, in the non-reduced CE-SDS method, a test sample is mixed with an SDS sample buffer and iodoacetamide. Thereafter, the mixture may be incubated at 68-72 ℃ for about 10-15 minutes, cooled to room temperature and the centrifuged supernatant used for analysis. And detecting the migration of the protein by adopting an ultraviolet detector to obtain an electrophoresis spectrogram. Antibody preparation purity can be calculated as the percentage of the peak area of the IgG main peak to the sum of all peak areas. For further description of the CE-SDS method, see, e.g., Richard R, et al, Application of CE SDS gel in reduction of biopharmaceutical anti-basic products, electrophosphoresis, 2008,29, 3612-3620.
Size exclusion high performance liquid chromatography, SEC-HPLC, is another important method for monoclonal antibody standardization and quality control. The method mainly separates molecules according to the size or the hydrodynamic radius difference of the molecules. By SEC-HPLC, antibodies can be isolated in three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram that is the sum of all peak areas. By SEC-HPLC, the percentage of antibody monomer in the formulated product can be measured, giving information on the content of soluble aggregates and shears. For further description of the SEC-HPLC method, see, e.g., J.Pharm.Scien.,83:1645-1650, (1994); pharm. Res.,11:485 (1994); J.pharm.Bio.anal.,15:1928 (1997); J.pharm.Bio.anal.,14:1133-1140 (1986). Furthermore, see, for example, R.Yang et al, High resolution section of recombinant monoclonal antibodies by size exclusion ultra-High performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and biological Analysis (2015), http:// dx.doi.org/10.1016/j.jpba.2015.02.032; and Alexandre Goyon et al, Protocols for the analytical characterization of thermal monoclonal antibodies.I-Non-condensing chromatographic techniques, Journal of Chromatography, http:// dx. doi. org/10.1016/j. jchromatography.2017.05.010.
Imaging capillary isoelectric focusing electrophoresis (iCIEF) can be used to analyze charge heterogeneity of monoclonal antibodies. The method can provide a quantitative distribution of charge variants. The iCIEF accomplishes the goal of molecular separation based on the difference in the charge of the molecule in the pH gradient (apparent pI value). In iCIEF, the separation column is typically a short capillary (e.g., a 5cm long, 100 μm inner diameter silica capillary), the protein is focused in a capillary column at high voltage, and the focus is monitored online in real time by a full-column imaging detection system operating at 280 nM. One advantage of this technique is that various charge variants of an antibody sample can be simultaneously recorded by the full column detection system. Generally, in icIEF, a sample is mixed with urea and icIEF buffer, wherein the buffer contains methylcellulose, pI molecular weight standards, and ampholytes. Spectra of focused mAb charge variants can then be obtained on an iCIEF analyzer, such as an iCE280 analyzer (Protein Simple, Santa Clara, Calif.), using an iCIEF column, such as a ProtionSimple assembled iCIEF column, and measuring the absorbance at 280nm after the sample has been focused for a certain time. In the iCEIF spectrum, protein-related peaks eluting before the main peak (i.e., the principal component) are classified as acidic components; in contrast, protein-related peaks that elute after the main peak are classified as basic components. The relative amounts of the main component, acidic component and basic component can be expressed as a percentage of the total peak area. For further description of iCIEF, see, for example, Salas-Solano O et al, Robustness of iCIEF methodology for the analysis of monoclonal antibodies, an intercalary study, J Sep Sci.2012 Nov; 35(22) 3124-9.doi 10.1002/jssc.201200633.Epub 2012 Oct 15; and Dada OO et al, chromatography of acidic and basic variants of IgG1 thermal monoclonal antibodies based on non-condensing IEF fractionation, electrophosphoresis.2015 Nov; 36(21-22) 2695-2702.doi 10.1002/elps.201500219.Epub 2015 Sep 18.
Charge variants of the antibodies in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, peaks eluting from the CEX-HPLC column earlier than the retention time of the main peak are labeled as "acidic peaks", while those eluting from the CEX-HPLC column later than the retention time of the main peak are labeled as "basic peaks".
Accelerated stability studies can be used to examine the stability properties of products, facilitating the screening of stable pharmaceutical formulation formats. For example, a sample of the formulation may be placed at an elevated temperature, e.g., about 40 ℃. + -. 2 ℃ and 25 ℃. + -. 2 ℃ for accelerated stability studies. The detection indices may include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).
In addition, the efficacy or biological activity of the antibody can be detected. For example, the ability of an antibody in a formulation to bind to its antigenic molecules (CD47 molecule and PD-L1 molecule) can be tested. Various methods are known to those skilled in the art for quantifying the specific binding of an antibody to an antigen, such as immunoassay tests, ELISA, and the like.
The anti-CD 47/PD-L1 bispecific antibody protein formulations of the present invention are stable. In one embodiment, the anti-CD 47/PD-L1 bispecific antibody protein purity in an antibody formulation of the invention is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more after storage at about 25 ℃, 37 ℃, 40 ℃, or 45 ℃ for at least 1 month or 2 months, e.g., after storage at 40 ℃ ± 2 ℃ for 1 month, as determined by size exclusion chromatography or by non-reduced CS-SDS. In one embodiment, at least 50%, preferably at least 55% of the anti-CD 47/PD-L1 bispecific antibody protein in an antibody formulation of the invention is in a non-basic and non-acidic form (i.e., the main peak or main charge form) after storage at about 25 ℃, 37 ℃, 40 ℃, or 45 ℃ for at least 1 month or 2 months, e.g., after storage at 40 ℃ ± 2 ℃ for 1 month, as determined by the CEX-HPLC method.
Use of the formulation
The antibody preparation of the invention comprising an anti-CD 47/PD-L1 bispecific antibody protein of the invention may be used for the treatment, prevention or delay of various diseases or disorders associated with the sirpa/CD 47 signaling pathway and/or with the PD1/PD-L1 signaling pathway. "a disease or disorder associated with the sirpa/CD 47 signaling pathway" and/or "a disease or disorder associated with the PD1/PD-L1 signaling pathway" herein refers to a disease or disorder that can be treated (e.g., ameliorated) or prevented with an anti-CD 47/PD-L1 bispecific antibody protein formulation of the invention. Any disease or disorder that may benefit from treatment with an antibody formulation of the invention is suitable for use in the invention.
In one aspect, the formulations of the invention comprising an anti-CD 47/PD-L1 bispecific antibody protein can be used to prevent or treat various hematological disorders and solid tumors in a subject, including but not limited to Acute Myeloid Leukemia (AML), chronic myeloid leukemia, Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), lymphoma, breast cancer, gastric cancer, lung cancer, esophageal cancer, intestinal cancer, ovarian cancer, cervical cancer, renal cancer, pancreatic cancer, bladder cancer, glioma, melanoma, and other solid tumors. In addition, human stem cell engraftment in NOD mouse lines can be enhanced by blocking the sirpa/CD 47 signaling pathway (WO 2009/046541), and thus, the formulations of the invention comprising an anti-CD 47/PD-L1 bispecific antibody protein also have potential benefits for use in human stem cell transplantation.
In another aspect, the formulations of the invention comprising an anti-CD 47/PD-L1 bispecific antibody protein can be used to treat, prevent or diagnose autoimmune and inflammatory disorders mediated by sirpa + cells, e.g., allergic asthma or ulcerative colitis, in a subject. These conditions include acute and chronic inflammatory conditions, allergic and allergic diseases, autoimmune diseases, ischemic conditions, severe infections, and rejection of cell or tissue or organ transplants, including non-human tissue transplant (xenograft) rejection, and the like.
The invention also provides the use of a formulation of the invention in the manufacture of a medicament for delivering an anti-CD 47/PD-L1 bispecific antibody protein to a mammal, or for treating, preventing or ameliorating one or more of the diseases and disorders described above. Preferably, the mammal is a human.
The antibody formulations of the invention can be administered to a subject or patient in a variety of ways. For example, administration may be by infusion or by syringe. Accordingly, in one aspect, the invention provides a delivery device (e.g., a syringe) comprising an antibody formulation of the invention (e.g., a pre-filled syringe). The patient will receive an effective amount of the anti-CD 47/PD-L1 bispecific antibody protein as the main active ingredient, i.e. an amount sufficient to treat, ameliorate or prevent the disease or disorder of interest.
The therapeutic effect may include a reduction in physiological symptoms. The optimal effective amount and concentration of antibody for any particular subject will depend upon a variety of factors including the age, weight, health and/or sex of the patient, the nature and extent of the disease, the activity of the particular antibody, its clearance by the body, and also includes any possible other treatments administered in combination with the antibody preparation. For a particular situation, the effective amount delivered may be determined within the judgment of the clinician. Depending on the indication to be treated, an effective dose may be from about 0.005mg/kg body weight to about 50mg/kg body weight, or from about 0.1mg/kg body weight to about 20mg/kg body weight. In this regard, the use of known antibody-based drugs may provide some guidance. The dosage may be a single dose schedule or a multiple dose schedule.
The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.
Acronym descriptions
CE-SDS: sodium dodecyl sulfate capillary gel electrophoresis
CEX-HPLC: cation exchange high performance liquid chromatography
ELISA: enzyme-linked immunosorbent assay
FLD-HPLC: fluorescence detection-high performance liquid chromatography
And iCIEF: imaging capillary isoelectric focusing electrophoresis
SEC-HPLC: size exclusion high performance liquid chromatography
ExamplesIn order to develop a preparation prescription of the recombinant anti-differentiation antigen cluster 47(CD47) and anti-programmed death ligand 1(PD-L1) bispecific antibody injection for long-term stable storage and ensure that the quality of a product is controllable in the period of validity (at least 24 months), a prescription screening test is designed and the influence of different auxiliary materials on the stability of the anti-CD 47/PD-L1 bispecific antibody preparation is examined. The materials and methods used for the tests were as follows:
materials and methods
1.1. Materials used in the formulation study of the present invention
Note: N/A indicates "Not applicable" (Not applicable).
1.2. The apparatus used in the formulation research of the present invention
1.3. Detection item and detection method for stability of preparation
The following items were tested for antibody formulations: (1) detecting the appearance and the presence of visible foreign matter; (2) determining the protein content of the formulation by ultraviolet method (UV method); (3) the purity of the antibody preparation, expressed as the area of the monomer as a percentage of the sum of all peak areas, is determined by size exclusion chromatography, e.g., size-exclusion high performance liquid chromatography (SEC-HPLC); (4) the purity of the antibody preparation was determined by reduced sodium dodecyl sulfate capillary electrophoresis (reduced CE-SDS) and/or non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS), expressed as the percentage of the area of the monomer to the sum of all peak areas; (5) determining the charge variants in the antibody preparation by imaging capillary isoelectric focusing electrophoresis (iCIEF method), expressed as a percentage of the main component, the acidic component and the basic component; (6) the relative binding activity of the anti-CD 47/PD-L1 bispecific antibody in the antibody preparation to the CD47 antigen and the PD-L1 antigen was determined by immunoassay, e.g., direct ELISA.
Detection of visible foreign matter
According to the national pharmacopoeia committee, the pharmacopoeia of the people's republic of China (2015 edition, 0904 "method for inspecting visible foreign matter" in general rules of four parts), Beijing, the pharmaceutical science and technology publisher of China, 2015, a clarity detector (Tianjin Daitan production, type YB-2) is used for inspecting visible foreign matters in the sample.
Protein content determination
The protein content in the sample was measured using an ultraviolet spectrophotometer (model UV-1800, Shimadzu, Japan).
Purity (SEC-HPLC method)
Separating with size exclusion chromatography column, wherein the mobile phase is phosphate buffer solution (3.12 g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine are weighed, the pH is adjusted to 6.8 by hydrochloric acid after the ultrapure water is dissolved, and the volume is adjusted to 1000ml), and the column protective solution is 0.05% (w/v) NaN3The sample amount is 50 mul, the flow rate is 0.5 ml/min, the collection time is 30min, the column temperature is 25 ℃, and the detection wavelength is 280 nm. A sample to be tested is diluted to 2mg/ml with ultrapure water to be used as a test solution. The preparation buffer was diluted in the same manner as described above to prepare a blank solution. 50 mul of each of the blank solution and the sample solution was injected into a liquid chromatograph, and the detection was started.
Purity (reduced CE-SDS method)
And (3) detecting by adopting a capillary gel electrophoresis method. The capillary tube is an uncoated capillary tube, the inner diameter of the capillary tube is 50 mu m, the total length of the capillary tube is 30.2cm, and the effective length of the capillary tube is 20.2 cm. Before electrophoresis, the capillary column was washed with 0.1mol/L NaOH, 0.1mol/L HCl, ultrapure water, and electrophoresis gel at 70 psi. Diluting a sample to be tested to 2.0mg/mL with a proper amount of ultrapure water, putting 50 mu l of the diluted sample into a 1.5mL centrifuge tube, respectively adding 45 mu l of sample buffer solution with pH6.5 (0.32 g of citric acid monohydrate and 2.45g of disodium hydrogen phosphate dodecahydrate are weighed and dissolved in 45mL of ultrapure water, fixing the volume to 50mL, preparing a citric acid-phosphate buffer solution, precisely weighing 200 mu l of the buffer solution, adding 80 mu l of 10% (w/v) sodium dodecyl sulfate solution, adding 1mL of water, uniformly mixing, obtaining 1 mu l of internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, product number: 390953) and 5 mu l of beta-mercaptoethanol, heating at 70 +/-2 ℃ for 10 +/-2 minutes after fully mixing, cooling to room temperature, and transferring to a sample bottle as a sample solution. The same volume of the buffer solution was used as the sample, and the same procedure was followed to obtain a blank solution. Sample introduction conditions of the sample are as follows: -5kV 20 seconds; separation voltage: 15kV 35 min. The capillary column temperature was controlled at 25 ℃ and the detection wavelength was 220 nm.
Purity (non-reduced CE-SDS method)
And (3) detecting by adopting a capillary gel electrophoresis method. The capillary tube is an uncoated capillary tube, the inner diameter of the capillary tube is 50 mu m, the total length of the capillary tube is 30.2cm, and the effective length of the capillary tube is 20.2 cm. Before electrophoresis, the capillary column was washed with 0.1mol/L NaOH, 0.1mol/L HCl, ultrapure water, and electrophoresis gel at 70 psi. Diluting a sample to be detected to 2.0mg/ml by using a proper amount of ultrapure water, taking 50 mu l of the diluted sample into a 1.5ml centrifugal tube, to each of them, 45. mu.l of a sample buffer solution having pH6.5 (0.32 g of citric acid monohydrate and 2.45g of disodium hydrogen phosphate dodecahydrate were weighed, dissolved in 45mL of ultrapure water, and the volume was adjusted to 50mL to obtain a citric acid-phosphate buffer solution, 200. mu.l of this buffer solution was precisely measured, 80. mu.l of a 10% (w/v) sodium dodecylsulfate solution was added, water was added to 1mL, and the mixture was mixed, 1. mu.l of an internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, cat # 390953) and 5. mu.l of a 250mmol/L NEM solution (62 mg of N-ethylmaleimide was weighed, dissolved in 2mL of ultrapure water) were mixed well, heated at 70. + -. 2 ℃ for 10. + -. 2 minutes, cooled to room temperature, and transferred to a sample bottle as a sample solution. The same volume of the buffer solution was used as the sample, and the same procedure was followed to obtain a blank solution. Sample introduction conditions of the sample are as follows: -5kV 20 seconds; separation voltage: 15kV 35 min. The capillary column temperature was controlled at 25 ℃ and the detection wavelength was 220 nm.
Charge variants (iCIEF method)
Detecting by imaging capillary isoelectric focusing electrophoresis (iCIEF method). The inner diameter of the capillary tube was 100 μm, and the total length was 5 cm. The capillary column was rinsed with 0.5% methylcellulose solution (hereinafter also abbreviated as MC solution) and ultrapure water, respectively, before electrophoresis. And (3) adopting a vacuum sample injection method for sample injection for 55 seconds, wherein the pre-focusing voltage and time are 1.5kV for 1 minute, the focusing voltage and time are 3kV for 8 minutes, the sample injection time is 55 seconds, the temperature of a sample plate is 10 ℃, the temperature of a capillary column is room temperature, and the detection wavelength is 280 nm. The Cathodic Stabilizer (Cathodic Stabilizer) is 500mmol/L arginine solution, the Anodic Stabilizer (Andhodic Stabilizer) is 200mmol/L iminodiacetic acid, 3mol/L urea improves protein solubility, and 0.5% MC solution reduces adhesion between protein and capillary. Diluting the sample with water to 0.5mg/ml, taking 20 μ l of the diluted sample solution, adding 83 μ l of the premixed solution, and mixing well to obtain the sample solution to be detected. The preparation buffer solution is used for preparing a blank solution by the same method.
Relative binding Activity (direct ELISA method)
Streptavidin (Thermo, cat # 21125) was diluted with 1 XPBS to 1. mu.g/ml, 100. mu.l/well and coated on 96-well microplate at 37 ℃ for 2 h. After washing the plates, blocking solution (5% FBS, 300. mu.l/well) was added and blocked for 2h at 37 ℃. Biotinylated antigen (for detection of the relative binding activity of anti-CD 47 to CD47 of the anti-CD 47/PD-L1 bispecific antibody, human CD47 protein (His-tag) from Sino biologicals of Beijing was used, cat # 12283-H08H-200; for detection of the relative binding activity of anti-PD-L1 to PD-L1 of the anti-CD 47/PD-L1 bispecific antibody, recombinant human PDL1/CD274 protein from ACRO BIOSYSTEM, cat # PD1-H5229-1MG) was used at 0.5. mu.g/ml, 100. mu.l/well, coated on 96-well enzyme plates at 37 ℃ for 0.5H, using 1 XPBS. 100 u L/hole with 2% FBS diluted anti-CD 47/PD-L1 bispecific antibody to 40 u g/ml, 4 times gradient dilution to the 12 th concentration (0.01 ~ 10000 ng/ml). The test sample diluted in the gradient was added to the washed microplate at 100. mu.l/well, and incubated at 37 ℃ for 30min in a constant temperature incubator. After washing the plate, HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, cat # A80-104P, USA) diluted with 2% FBS was added as a secondary antibody (30000-fold dilution, 100. mu.l/well) for 20min at 37 ℃. After washing the plate, 100. mu.l of TMB developing solution was added, and after 10min of development, 100. mu.l of 1mol/L H was added to each well2SO4The reaction was terminated. The OD value at 450nm was measured using 620nm as a reference wavelength. The concentration value of each concentration gradient sample is taken as an abscissa, the OD450nm-OD620nm value of each gradient sample is taken as an ordinate, and the EC is calculated by Prism four-parameter fitting50Reflecting the binding activity of the antibody to each antigen.
Example 1 preparation and purification of an anti-CD 47/PD-L1 bispecific antibody
The anti-CD 47/PD-L1 bispecific antibody Kh2NF-PC was recombinantly expressed and purified in HEK293 cells (purchased from INVITROGEN) according to the teachings of PCT/CN 2018/123886. The anti-CD 47/PD-L1 bispecific antibody Kh2NF-PC antibody consists of 3 polypeptide chains, and each polypeptide chain has the following amino acid sequences from N end to C end:
peptide chain # 1:
wherein the peptide chain #1 comprises the following VH amino acid sequence derived from anti-CD 47 antibody ADI 29341:
the following CH1 amino acid sequence derived from human IgG1 at the C-terminus of the VH amino acid sequence:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 6); and
the amino acid sequence of the following Fc region derived from human IgG1 at the C-terminus of the CH1 amino acid sequence:
peptide chain # 2:
wherein the peptide chain #2 comprises the following VL amino acid sequence derived from anti-CD 47 antibody ADI 29341:
DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVSFPITFGGGTKVEIK (SEQ ID NO: 9); and
the following human kappa light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence:
peptide chain #3
Wherein the peptide chain #3 comprises the following first and second anti-PD-L1 VHH amino acid sequences:
a linker peptide amino acid sequence between the first and second anti-PD-L1 VHH amino acid sequences: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20); and
the following amino acid sequence of the Fc region derived from human IgG1 at the C-terminal end of the amino acid sequence of the second anti-PD-L1 VHH:
example 2 one of the tests for the influence of pH on the stability of the formulations
This example investigates the stability of formulations comprising anti-CD 47/PD-L1 bispecific antibodies at pH 5.0 to 6.5. A total of 4 pH values were designed, 5.0, 5.5, 6.0 and 6.5 respectively.
2.1 Experimental procedures
10mM histidine, 5% (w/v) sorbitol buffer, pH adjusted to 5.0, 5.5, 6.0 and 6.5 with dilute hydrochloric acid, purified anti-CD 47/PD-L1 bispecific antibody Kh2NF-PC protein (7.3mg/ml) was ultrafiltered and replaced into the different pH solutions. Adjusting the bispecific antibody protein content in the sample to about 100.0mg/ml after the displacement is complete; adding polysorbate 80 to make the final concentration 0.70 mg/ml; filtering, subpackaging into 2R penicillin bottles, plugging and capping. The stability of each sample was investigated at 40 ℃. + -. 2 ℃ and the specific experimental protocol is shown in Table 1.
TABLE 1 Experimental protocol
Note: (1) x represents the sampling at this time point. (2) And after sampling at the time point, putting the obtained sample into an ultra-low temperature refrigerator for freezing and storing for inspection, and thawing and inspecting according to requirements.
2.2 criteria of judgment
According to the knowledge of the product and the precision of the instrument and the method, a determination standard that the quality of the sample detection index value is unchanged from the initial value is set to judge whether the sample is changed, and the table 2 shows the specific.
TABLE 2 judgment criteria for unchanged mass
2.3 Experimental results of one of the prescription screening tests
(1) Appearance and visible foreign matter
After the sample is placed for one month at the temperature of 40 +/-2 ℃, the samples with the pH of 5.0, the pH of 5.5 and the pH of 6.0 are turbid and precipitate to different degrees in appearance; only the pH6.5 samples were acceptable for both appearance and visible foreign matter.
(2) Protein content
The results of the protein content measurements for each sample after various times of incubation at 40 ℃. + -. 2 ℃ at pH 5.0, 5.5, 6.0 and 6.5 are shown in Table 3. The results show that the protein content of the sample with the pH value of 6.5 is not changed significantly when the sample is placed at the temperature of 40 +/-2 ℃ for 1 month.
TABLE 3 protein content of the samples after different periods of time at pH 5.0, 5.5, 6.0 and 6.5 at 40 ℃. + -. 2 ℃ (UV method, mg/ml)
Note: N/A indicates that the sample has failed appearance and is not tested.
(3) Purity of
Protein purity of each sample was determined by SEC-HPLC after standing at pH 5.0, 5.5, 6.0 and 6.5 at 40 ℃. + -. 2 ℃ for various periods of time. The results are shown in Table 4. The results show that the protein purity of the sample with pH6.5 value is reduced by 4.1% compared with the sample with 0 day when the sample is examined for 1 month under the condition of 40 ℃ +/-2 ℃.
TABLE 4 protein purity (%)
Note: N/A indicates that the sample has failed appearance and is not tested.
After leaving at pH 5.0, 5.5, 6.0 and 6.5 at 40. + -. 2 ℃ for various periods of time, the protein purity of each sample was determined by the non-reduced CE-SDS method and the reduced CE-SDS method, respectively. The results are shown in tables 5 and 6. The results show that the protein purity of the sample with pH6.5 value is reduced by 7.7% and 3.7% respectively compared with the sample with 0 day when the sample is examined for 1 month under the condition of 40 +/-2 ℃.
TABLE 5 protein purity (%)
Note: N/A indicates that the sample has failed appearance and is not tested.
TABLE 6 protein purity (%)
Note: N/A indicates that the sample has failed appearance and is not tested.
(4) Charge variants
After standing at pH 5.0, 5.5, 6.0 and 6.5 at 40 ℃. + -. 2 ℃ for various periods of time, the charge variants of each sample were determined by the iCIEF method. The results are shown in Table 7. The results show that the main component and the acid component of the sample with the pH of 6.5 are obviously changed after being examined for 1 month under the condition of 40 +/-2 ℃. Compared with the 0-day sample, the acidic component increased from 36.6% to 60.1%, which is increased by 23.5%; the main component is reduced from 62.5 percent to 39.1 percent, and is reduced by 23.4 percent; the alkaline component did not change significantly.
TABLE 7 Charge variants (%)
Note: N/A indicates that the sample has failed appearance and is not tested.
(5) Relative binding Activity
The relative binding activity of each sample was determined by direct ELISA after standing at pH 5.0, 5.5, 6.0 and 6.5 at 40 ℃. + -. 2 ℃ for various periods of time. The results are shown in Table 8. The results show that the relative binding activity of the anti-CD 47 end and the anti-PD-L1 end of the protein on CD47 and PD-L1 is unchanged in the sample with pH6.5 and under the condition of 40 +/-2 ℃ for 1 month.
TABLE 8 relative binding Activity of samples determined by direct ELISA (%)
Note: N/A1Indicating that the appearance of the sample is unqualified and is not detected; N/A2Indicating that the detection item is not set.
The above experimental results show that the anti-CD 47/PD-L1 bispecific antibody protein (e.g., Kh2NF-PC protein) is stable at pH6.5 for formulations at pH 5.0, 5.5, 6.0, and 6.5. Further experiments were performed in order to investigate the stability of the formulations in the pH range around pH 6.5.
Example 3 second test for pH Effect on formulation stability
This example examined the effect of pH 6.2 to 7.0 on protein stability in an anti-CD 47/PD-L1 bispecific antibody formulation, with a total of 5 pH values designed, 6.2, 6.4, 6.5, 6.8 and 7.0, respectively.
3.1 Experimental procedures
The specific procedure was the same as in "2.1 Experimental step" in example 2, except that the pH was different.
3.2 criteria of judgment
See table 2 in example 2.
3.3 results of the experiment
(1) Appearance and visible foreign matter
After being placed for one month at the temperature of 40 +/-2 ℃, the sample with the pH of 6.2 is milky in appearance; the samples at pH 6.4, 6.5, 6.8 and 7.0 all had acceptable appearance and visible foreign matter.
(2) Protein content
The results of the protein content measurements for each sample after various times of incubation at 40 ℃. + -. 2 ℃ at pH 6.4, 6.5, 6.8 and 7.0 are shown in Table 9. The results show that the protein content of the samples of pH 6.4, 6.5, 6.8 and 7.0 is not changed significantly when the samples are placed at 40 +/-2 ℃ for 1 month.
TABLE 9 protein content of the samples after different periods of time at 40 ℃. + -. 2 ℃ at pH 6.4, 6.5, 6.8 and 7.0 (UV method, mg/ml)
Note: N/A indicates that the detection item is not set.
(3) Purity of
Protein purity of each sample was determined by SEC-HPLC after standing at pH 6.4, 6.5, 6.8 and 7.0 at 40 ℃. + -. 2 ℃ for various periods of time. The results are shown in Table 10, and the trend of purity is shown in FIG. 2. The results show that the purity of the samples at pH 6.4, 6.5, 6.8 and 7.0 all decreased by 3.0%, 3.7%, 4.9% and 5.6% compared to 0 days, respectively, when examined at 40 ℃. + -. 2 ℃ for 1 month.
TABLE 10 protein purity (%)
After leaving at pH 6.4, 6.5, 6.8 and 7.0 at 40 ℃. + -. 2 ℃ for various times, the protein purity of each sample was determined by the non-reduced CE-SDS method, respectively. The results are shown in Table 11, and the trend of purity is shown in FIG. 3. The results show that the purity of the samples at pH 6.4, 6.5, 6.8 and 7.0 all decreased by 7.0%, 6.1%, 6.7% and 7.3% compared to 0 days, respectively, when examined at 40 ℃. + -. 2 ℃ for 1 month.
TABLE 11 protein purity (%)
Note: N/A indicates that the detection item is not set.
(4) Charge variants
After standing at pH 6.4, 6.5, 6.8 and 7.0 at 40 ℃. + -. 2 ℃ for various periods of time, the charge variants of each sample were determined by the iCIEF method. The results are shown in Table 12, and the trend of purity is shown in FIG. 4. The result shows that the main components and the acid components of the samples with different pH values are changed after being examined for 1 month at the temperature of 40 +/-2 ℃. The higher the pH, the faster the major component will fall and the faster the acidic component will rise.
TABLE 12 Charge variants (%)
Note: N/A indicates that the detection item is not set.
(5) Relative binding Activity
The relative binding activity of each sample was determined by direct ELISA after standing at pH 6.4, 6.5, 6.8 and 7.0 at 40 ℃. + -. 2 ℃ for various periods of time. The results are shown in Table 13. The results show that the relative binding activity of the anti-CD 47 and anti-PD-L1 terminals on CD47 and PD-L1 of the proteins in the samples with pH 6.4, pH6.5 and pH 7.0 is not changed obviously when the samples are examined for 1 month at 40 ℃ +/-2 ℃.
TABLE 13 relative binding Activity of samples determined by direct ELISA (%)
Note: N/A indicates that the detection item is not set.
The test results of pH effect on formulation stability of examples 2 and 3 show that the anti-CD 47/PD-L1 bispecific antibody (e.g., Kh2NF-PC) protein is placed at pH 5.0-6.2 at 40 ℃ + -2 ℃ for one month, and the appearance of the sample is cloudy or milky white precipitate with time; and when the sample is placed at the pH of 6.4-7.0 and the temperature of 40 +/-2 ℃ for one month, the appearance and visible foreign matters of the sample are qualified, the protein content is not changed obviously, and the relative binding activity to CD47 and PD-L1 is not changed obviously. Thus, in the examples that follow, the subsequent experiments were carried out with a pH of 6.5 selected from pH 6.4-7.0.
Example 4 prescription screening test
4.1 stabilizer screening test
Different stabilizers were investigated: the effect of sorbitol, sucrose, trehalose, arginine hydrochloride, etc. on the stability of bispecific antibody formulations comprising anti-CD 47/PD-L1.
4.1.1 stabilizer screening assay procedure
A total of 5 prescriptions are designed and the detailed prescription information is shown in table 14. Each of the prescribed buffers was prepared according to Table 14, and the anti-CD 47/PD-L1 bispecific antibody Kh2NF-PC protein (3.6mg/ml) was ultrafiltered and replaced into the respective prescribed solutions. After the replacement is completed, adjusting the protein content of each prescription to about 100.0 mg/ml; adding polysorbate 80 to make the final concentration of polysorbate 80 be 0.20 mg/ml; filtering, subpackaging into penicillin bottles, plugging and capping. The stability of each sample was examined at 40 ℃. + -. 2 ℃ and the specific protocol is shown in Table 15. The detection indexes are appearance, visible foreign matter, protein content, purity (SEC-HPLC method) and charge variant (iCIEF method).
TABLE 14 information Table of alternative recipes for stabilizer screening test
| Serial number | Prescription information |
| Prescription 1 | 20mM histidine, 5% sorbitol, 0.02% polysorbate 80, pH 6.0 |
| Prescription 2 | 20mM histidine, 5% sorbitol, 0.02% polysorbate 80, pH6.5 |
| Prescription 3 | 20mM histidine, 8% trehalose, 0.02% polysorbate 80, pH6.5 |
| Prescription 4 | 20mM histidine, 180mM arginine hydrochloride, 0.02% polysorbate 80, pH6.5 |
| Prescription 5 | 20mM histidine, 4% sucrose, 100mM arginine hydrochloride, 0.02% polysorbate 80, pH6.5 |
Note: in the table,% means% w/v, as follows.
TABLE 15 stability test protocol
Note: (1) x represents the point sample. (2) After the sampling at the time points, the obtained samples are put into an ultra-low temperature refrigerator for cryopreservation to be detected, and are frozen and delivered for inspection according to the requirements.
4.1.2 criteria
See table 2 in example 2 for criteria for the determination.
4.1.3 stabilizer screening test
(1) Appearance, visible foreign matter
Observing at 40 +/-2 ℃ for 4 weeks, and observing for a week according to the formula 1 to obtain turbidity or precipitates; the appearance and visible foreign matters of other prescription samples are qualified.
(2) Protein content
The results of 4 weeks at 40 ℃. + -. 2 ℃ were found in Table 16. As can be seen from Table 16, the protein content of recipe 2, recipe 3, recipe 4, and recipe 5 did not change when left at 40 ℃. + -. 2 ℃ for 4 weeks.
TABLE 16 protein content results of stabilizer screening test (UV method, mg/ml)
Note: N/A indicates that the sample has failed appearance and is not tested.
(3) Purity of
Purity (SEC-HPLC method): the results of 4 weeks observation at 40 ℃. + -. 2 ℃ are shown in Table 17, and the trend of purity is shown in FIG. 5. The results show that the purity of the formula 2, formula 3, formula 4 and formula 5 samples is reduced by 2.6%, 3.0%, 0.7% and 0.7% respectively compared with the purity of the 0-day sample when the samples are examined for 4 weeks at 40 +/-2 ℃.
TABLE 17 purity results of the stabilizer screening test (SEC-HPLC method,%)
Note: N/A indicates that the sample has failed appearance and is not tested.
Purity (non-reduced CE-SDS method): the results of 4 weeks observation at 40 ℃. + -. 2 ℃ are shown in Table 18, and the trend of purity is shown in FIG. 6. The result shows that the purity of each sample is reduced after 4 weeks of investigation at 40 +/-2 ℃; recipe 2, recipe 3, recipe 4, and recipe 5 decreased by 6.8%, 7.2%, 9.1%, and 9.2%, respectively, compared to the 0-day sample purity.
TABLE 18 purity results of the stabilizer screening test (non-reduced CE-SDS method,%)
Note: N/A indicates that the sample has failed appearance and has not been tested
(4) Charge variants (iCIEF method)
The results of 4 weeks of charge variation observed at 40 ℃. + -. 2 ℃ are shown in Table 19, and the trend of charge variation of the major components is shown in FIG. 7.
The result shows that the main component and the acidic component of the charge variant in each part are obviously changed after 4 weeks of examination at 40 +/-2 ℃, the main component is reduced, the acidic component is increased, and the change trends are basically consistent.
TABLE 19 Charge variant results of stabilizer screening test (iCIEF method,%)
Note: N/A indicates that the sample has failed appearance and has not been tested
The results of the stabilizer screening test show that when the formula 2, the formula 3, the formula 4 and the formula 5 are placed at 40 +/-2 ℃ for 4 weeks, the appearance and visible foreign matters of the sample are qualified, the protein content is unchanged, the purity of the sample is slightly reduced, and the formula 4 and the formula 5 have obvious advantages in the aspects of purity (SEC-HPLC method) and charge variants (iCIEF method).
4.2 osmotic pressure test
4.2.1 osmotic pressure test procedure
Osmolarity was determined on 0 hour (T0) samples of formula 4 and formula 5 using a multichannel osmometer. Each set of samples was measured 2 times and the results averaged.
4.2.2 osmotic pressure test results
The results of the osmolarity measurements on the 0 hour (T0) samples of formula 4 and formula 5 are shown in Table 20.
TABLE 20 osmotic pressure measurement results
Since the human plasma osmolality is about 285-310 mOsmol/kg, the osmolality of formula 4 and formula 5 are within an acceptable range for the drug formulation. In addition, the formulation of formula 4 is preferably used in view of the fact that the osmotic pressure of formula 4 is closer to that of human plasma.
Example 5: effect of Metal chelators on formulation stability
EDTA is a representative metal chelator. This example investigated the effect of EDTA on the stability of Kh2NF-PC protein, an anti-CD 47/PD-L1 bispecific antibody.
5.1 EDTA study protocol for formulation stability:
a total of 2 prescriptions were designed, with prescription 6 being a control without EDTA; recipe 7 is a group with a final concentration of 0.02mg/ml EDTA. The detailed prescription information is shown in table 21. Each prescription buffer was prepared according to table 21 and the anti-CD 47/PD-L1 bispecific antibody Kh2NF-PC protein was ultrafiltered and replaced into each prescription solution. After the replacement was complete, the protein content of each formulation was adjusted to about 100.0 mg/ml.
TABLE 21 prescription information Table of EDTA for formulation stability
The detailed experimental conditions and sampling schedule for the effect of EDTA on formulation stability are shown in table 22.
TABLE 22 protocol for EDTA evaluation of formulation stability
Note: (1) x represents the point sample. (2) After the sampling at the time points, the obtained samples are put into an ultra-low temperature refrigerator for cryopreservation to be detected, and are frozen and delivered for inspection according to the requirements.
5.2 results of the experiment
The observation was carried out at 40 ℃. + -. 2 ℃ for 4 weeks, and the results are shown in Table 23.
TABLE 23 Experimental results of EDTA on formulation stability
The results in Table 23 show that the charge variant main peak of formula 7 shows better superiority compared with that of formula 6 from the detection result of charge variant (CEX-HPLC method); from the results of the polysorbate 80(FLD-HPLC method) test, the polysorbate 80 content of formula 6 decreased with time; formula 7 is superior to formula 6 in that the metal chelating agent EDTA is added to inhibit degradation of polysorbate 80 due to metal ions. EDTA, as an example of a metal chelator, is capable of binding metal ions that inhibit degradation of polysorbate 80 from at least two sources. One is that some metal ions may be introduced in the whole production process of the protein, including cell culture, purification and other related operation steps, thereby causing the polysorbate 80 to be subjected to oxidative degradation under the coexistence of oxygen and the metal ions; secondly, some host cell proteins may remain in the protein in the prescription, related enzymes which degrade polysorbate 80 may exist in the hybrid proteins, and the enzymes need metal ions as cofactors to play a catalytic function, so that the metal chelating agent added in the prescription can inhibit the degradation of polysorbate 80 by combining the metal ions and improve the stability of the prescription.
Thus, the most preferred formulation protocol was determined to be: about 100.0mg/ml recombinant anti-differentiation antigen cluster 47(CD47) and anti-programmed death ligand 1(PD-L1) bispecific antibody, about 2.52mg/ml histidine, 0.79mg/ml histidine hydrochloride, 37.92mg/ml arginine hydrochloride, 0.50mg/ml polysorbate 80, 0.02mg/ml EDTA, pH 6.5.
Example 6 stability Studies of 500L preparation
A500L preparation was prepared using the formulation protocol of example 5 (about 100.0mg/ml recombinant anti-differentiation antigen cluster 47(CD47) and anti-programmed death ligand 1(PD-L1) bispecific antibody, about 2.52mg/ml histidine, 0.79mg/ml histidine hydrochloride, 37.92mg/ml arginine hydrochloride, 0.50mg/ml polysorbate 80, 0.02mg/ml EDTA, pH 6.5) for stability studies.
6.1 preparation to be investigated for stability and investigation protocol
500L of the following preparation was prepared: 101.8mg/ml recombinant anti-CD 47/PD-L1 bispecific antibody protein, 2.52mg/ml histidine, 0.79mg/ml histidine hydrochloride, 37.92mg/ml arginine hydrochloride, 0.50mg/ml polysorbate 80, 0.02mg/ml EDTA, pH6.5, for stability.
TABLE 24 protocol for investigating the stability of the preparations
TABLE 25 quality standards
Note: 1. the report data refers to that the acceptance range is not set for the detection, and the actually detected data is directly reported. The same applies below.
2. In the SEC-HPLC method, the main peak + aggregate + fragment is 100%, as long as the main peak satisfies the condition of 95% or more, and the remaining 5% or less is the amount of the fragment and the aggregate, and thus, the data of both the amounts of the fragment and the aggregate are given as the report data. The same applies below.
3. In the non-reduced CE-SDS method, the amount of fragments is given as the reported data, since the main peak + fragments are 100% and the remaining 10% or less is the amount of fragments as long as the main peak satisfies the condition of 90% or more. The same applies below.
4. In the reduced CE-SDS method, the heavy and light chain contents + the non-glycosylated heavy chain + the fragment were 100%, and as long as the heavy and light chain contents satisfied the condition of 90% or more, the remaining 10% or less was the amount of the non-glycosylated heavy chain and the fragment, and therefore, data of both the amount of the non-glycosylated heavy chain and the amount of the fragment were given as reported data, respectively. The same applies below.
5. In the CEX-HPLC method, the principal component + acidic component + basic component is 100%, and as long as the amount of the principal component satisfies the condition of 44.9% or more, the remaining 55.1% or less is the amount of the acidic component and the basic component, and therefore, data of both the amount of the acidic component and the amount of the basic component are given as reported data, respectively. The same applies below.
6. The provision is: the following general regulation of pharmacopoeia of the people's republic of china (2015 edition, three parts) for example 0904, "method for inspecting visible foreign matter" regulates the inspection of visible foreign matter.
6.2 stability test results
The results of stability studies for the 500L preparation are shown in the table below.
TABLE 26 Long-term stability study results (5 ℃. + -. 3 ℃)
Note: and 1, N/A indicates that detection is not performed, the corresponding indexes generally only detect the head end and the tail end, and if the point values of the head end and the tail end meet the requirements, the value of each point in the process is considered to meet the requirements.
2. In the reduced CE-SDS method, the main peak + NGHCCD47+ fragment is 100%, as long as the main peak satisfies the condition of more than or equal to 90%, the rest less than or equal to 10% is NGHCCD47Amount of chains and fragments, therefore, NGHCCD47The respective data for both the amount of fragments and the amount of fragments are given as report data.
As can be seen from the test results in table 26, the preparations met the quality standards after 6 months.
TABLE 27 accelerated stability study results (25 ℃. + -. 2 ℃/60% RH. + -. 5% RH)
Note: N/A represents that no detection exists, the corresponding indexes generally only detect the head end and the tail end, and if the point values of the head end and the tail end meet the requirements, the value of each point in the process is considered to meet the requirements.
As can be seen from the test results in table 27, in the accelerated stability test, each index of the preparation was satisfied after 6 months.
TABLE 28 forced conditions test results (40 ℃. + -. 2 ℃/75% RH. + -. 5% RH)
Note: N/A represents that no detection exists, the corresponding indexes generally only detect the head end and the tail end, and if the point values of the head end and the tail end meet the requirements, the value of each point in the process is considered to meet the requirements.
As can be seen from the test results in table 28, in the forced stability test, each index of the preparation was satisfactory after 8 weeks.
In summary, it was found through the above experiments that the formulation protocol of the present invention satisfies the stability requirements for the formulation in scale-up production.
Having described exemplary embodiments of the invention, it will be understood by those skilled in the art that the present disclosure is illustrative only, and various other substitutions, adaptations and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments set forth herein.